Anti-NGF antibodies and methods using same

ABSTRACT

The invention concerns anti-NGF antibodies (such as anti-NGF antagonist antibodies), and polynucleotides encoding the same. The invention further concerns use of such antibodies and/or polynucleotides in the treatment and/or prevention of pain, including post-surgical pain, rheumatoid arthritis pain, and osteoarthritis pain.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of provisionalpatent applications U.S. Ser. No. 60/436,905, filed Dec. 24, 2002; U.S.Ser. No. 60/443,522, filed Jan. 28, 2003; and U.S. Ser. No. 60/510,006,filed Oct. 8, 2003 all of which are incorporated herein by reference intheir entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with U.S. Government support underContract No. DAAD19-03-C-0006, awarded by DARPA. The U.S. Government mayhave certain rights in this invention.

FIELD OF THE INVENTION

[0003] The invention concerns anti-NGF antibodies (such as anti-NGFantagonist antibodies). The invention further concerns use of suchantibodies in the treatment and/or prevention of pain, includingpost-surgical pain, rheumatoid arthritis pain, and osteoarthritis pain.

BACKGROUND OF THE INVENTION

[0004] Nerve growth factor (NGF) was the first neurotrophin to beidentified, and its role in the development and survival of bothperipheral and central neurons has been well characterized. NGF has beenshown to be a critical survival and maintenance factor in thedevelopment of peripheral sympathetic and embryonic sensory neurons andof basal forebrain cholinergic neurons. Smeyne et al., Nature368:246-249 (1994) and Crowley et al., Cell 76:1001-1011 (1994). NGFup-regulates expression of neuropeptides in sensory neurons (Lindsay andHarmer, Nature 337:362-364 (1989)) and its activity is mediated throughtwo different membrane-bound receptors, the TrkA receptor and the p75common neurotrophin receptor (sometimes termed “high affinity” and “lowaffinity” NGF receptors, respectively). Chao et al., Science 232:518-521(1986). For review on NGF, see Huang et al., Annu. Rev. Neurosci.24:677-736 (2001); Bibel et al., Genes Dev. 14:2919-2937 (2000). Thecrystal structure of NGF and NGF in complex with the trkA receptor havebeen determined. See Nature 254:411 (1991); Nature 401:184-188 (1996).

[0005] Nerve growth factor (NGF) was the first neurotrophin to beidentified, and its role in the development and survival of bothperipheral and central neurons has been well characterized. NGF has beenshown to be a critical survival and maintenance factor in thedevelopement of peripheral sympathetic and embryonic sensory neurons andof basal forebrain cholinergic neurons (Smeyne, et al., Nature368:246-249 (1994) and Crowley, et al., Cell 76:1001-1011 (1994)). NGFupregulates expression of neuropeptides in sensory neurons (Lindsay, etal., Nature 337:362-364 (1989)), and its activity is mediated throughtwo different membrane-bound receptors, the TrkA tyrosine kinasereceptor and the p75 receptor which is structurally related to othermembers of the tumor necrosis factor receptor family (Chao, et al.,Science 232:518-521 (1986)).

[0006] In addition to its effects in the nervous system, NGF has beenincreasingly implicated in processes outside of the nervous system. Forexample, NGF has been shown to enhance vascular permeability (Otten, etal., Eur J Pharmacol. 106:199-201 (1984)), enhance T- and B-cell immuneresponses (Otten, et al., Proc. Natl. Acad. Sci. USA 86:10059-10063(1989)), induce lymphocyte differentiation and mast cell proliferationand cause the release of soluble biological signals from mast cells(Matsuda, et al., Proc. Natl. Acad. Sci. USA 85:6508-6512 (1988);Pearce, et al., J. Physiol. 372:379-393 (1986); Bischoff, et al., Blood79:2662-2669 (1992); Horigome, et al., J. Biol. Chem. 268:14881-14887(1993)). Although exogenously added NGF has been shown to be capable ofhaving all of these effects, it is important to note that it has onlyrarely been shown that endogenous NGF is important in any of theseprocesses in vivo (Torcia, et al., Cell. 85(3):345-56 (1996)).Therefore, it is not clear what that effect might be, if any, ofinhibiting the bioactivity of endogenous NGF.

[0007] NGF is produced by a number of cell types including mast cells(Leon, et al., Proc. Natl. Acad. Sci. USA 91:3739-3743 (1994)),B-lymphocytes (Torcia, et al., Cell 85:345-356 (1996), keratinocytes (DiMarco, et al., J. Biol. Chem. 268:22838-22846)), smooth muscle cells(Ueyama, et al., J. Hypertens. 11:1061-1065 (1993)), fibroblasts(Lindholm, et al., Eur. J. Neurosci. 2:795-801 (1990)), bronchialepithelial cells (Kassel, et al., Clin, Exp. Allergy 31:1432-40 (2001)),renal mesangial cells (Steiner, et al., Am. J. Physiol. 261:F792-798(1991)) and skeletal muscle myotubes (Schwartz, et al., J. Photochem.Photobiol. B66: 195-200 (2002)). NGF receptors have been found on avariety of cell types outside of the nervous system. For example, TrkAhas been found on human monocytes, T- and B-lymphocytes and mast cells.

[0008] An association between increased NGF levels and a variety ofinflammatory conditions has been observed in human patients as well asin several animal models. These include systemic lupus erythematosus(Bracci-Laudiero, et al., Neuroreport 4:563-565 (1993)), multiplesclerosis (Bracci-Laudiero, et al., Neurosci. Lett. 147:9-12 (1992)),psoriasis (Raychaudhuri, et al., Acta Derm. l'enereol. 78:84-86 (1998)),arthritis (Falcim, et al., Ann. Rheum. Dis. 55:745-748 (1996)),interstitital cystitis (Okragly, et al., J Urology 161:438-441 (1999))and asthma (Braun, et al., Eur. J. Immunol. 28:3240-3251 (1998)).

[0009] Consistently, an elevated level of NGF in peripheral tissues isassociated with hyperalgesia and inflammation and has been observed in anumber of forms of arthritis. The synovium of patients affected byrheumatoid arthritis expresses high levels of NGF while in non-inflamedsynovium NGF has been reported to be undetectable (Aloe, et al., Arch.Rheum. 35:351-355 (1992)). Similar results were seen in rats withexperimentally induced rheumatoid arthritis (Aloe, et al., Clin. Exp.Rheumatol. 10:203-204 (1992)). Elevated levels of NGF have been reportedin transgenic arthritic mice along with an increase in the number ofmast cells (Aloe, et al., Int. J. Tissue Reactions-Exp. Clin. Aspects15:139-143 (1993)). PCT Publication No. WO 02/096458 discloses use ofanti-NGF antibodies of certain properties in treating various NGFrelated disorders such as inflammatory condition (e.g., rheumatoidarthritis). It has been reported that a purified anti-NGF antibodyinjected into arthritic transgenic mice carrying the human tumornecrosis factor-α (TNF-α) gene caused reduction in the number of mastcells, as well as a decrease in histamine and substance P levels withinthe synovium of arthritis mice (Aloe et al., Rheumatol. Int. 14: 249-252(1995)). It has been shown that exogenous administration of a NGFantibody reduced the enhanced level of TNF-α occurring in arthritic mice(Manni et al., Rheumatol. Int. 18: 97-102 (1998)).

[0010] Also, increased expression of NGF and high affinity NGF receptor(TrkA) was observed in human osteoarthritis chondrocytes (Iannone etal., Rheumatology 41:1413-1418 (2002)).

[0011] Rodent anti-NGF antagonist antibodies have been reported. See,e.g., Hongo et al, Hybridoma (2000) 19(3):215-227; Ruberti et Al. (1993)Cell. Molec. Neurobiol. 13(5): 559-568. However, when rodent antibodiesare used therapeutically in humans, a human anti-murine antibodyresponse develops in significant numbers of treated individuals. Inaddition, effector functions of mouse antibodies have proven to be lessefficient in the human context. Thus, there is a serious need foranti-NGF antagonist antibodies, including humanized anti-NGF antagonistantibodies.

[0012] All references, publications, and patent applications disclosedherein are hereby incorporated by reference in their entirety.

BRIEF SUMMARY OF THE INVENTION

[0013] The invention disclosed herein concerns antibodies to nervegrowth factor.

[0014] In another aspect, the invention is a humanized and affinitymatured antibody, E3, which specifically binds human and rodent nervegrowth factor (“NGF”). The amino acid sequences of the heavy chain andlight chain variable regions of E3 are shown in FIGS. 1A (SEQ ID NO:1)and 1B (SEQ ID NO:2), respectively. The CDR portions of antibody E3(including Chothia and Kabat CDRs) are diagrammatically depicted inFIGS. 1A and 1B. The amino acid sequences of E3 heavy and light chains,and of the individual extended CDRs are also shown below (See, “antibodysequences”, below).

[0015] In another aspect, the invention is an antibody comprising afragment or a region of the antibody E3 (interchangeably termed “E3”herein). In one embodiment, the fragment is a light chain of theantibody E3 as shown in FIG. 1B. In another embodiment, the fragment isa heavy chain of the antibody E3 as shown in FIG. 1A. In yet anotherembodiment, the fragment contains one or more variable regions from alight chain and/or a heavy chain of the antibody E3. In yet anotherembodiment, the fragment contains one or more complementaritydetermining regions (CDRS) from a light chain and/or a heavy chain ofthe antibody E3 as shown in FIGS. 1A and 1B.

[0016] In another aspect, the invention is an antibody comprising alight chain that is encoded by a polynucleotide that is produced by ahost cell with a deposit number of ATCC No. PTA-4893 or ATCC No.PTA-4894. In another aspect, the invention is an antibody comprising aheavy chain that is encoded by a polynucleotide that is produced by ahost cell with a deposit number of ATCC No. PTA-4895. In another aspect,the invention is an antibody comprising (a) a light chain that isencoded by a polynucleotide that is produced by a host cell with adeposit number of ATCC No. PTA-4894 or ATCC No. PTA-4893; and (b) aheavy chain that is encoded by a polynucleotide that is produced by ahost cell with a deposit number of ATCC No. PTA-4895 (for convenienceherein, the polynucleotide(s) produced by a deposited host cell arereferred to as having a deposit number of ATCC NOs PTA-4894, PTA-4893and PTA-4895). In another aspect, the invention is an antibodycomprising a light chain variable region of a light chain that isencoded by a polynucleotide that is produced by a host cell with adeposit number of ATCC No. PTA-4894 or ATCC No. PTA-4893. In anotheraspect, the invention is an antibody comprising a heavy chain variableregion of a heavy chain that that is encoded by a polynucleotide that isproduced by a host cell with a deposit number of ATCC No. PTA-4895. Inanother aspect, the invention is an antibody comprising (a) a lightchain variable region of a light chain that is encoded by apolynucleotide that is produced by a host cell with a deposit number ofATCC No. PTA-4894 or ATCC No. PTA-4893, and (b) a heavy chain variableregion of a heavy chain that that is encoded by a polynucleotide that isproduced by a host cell with a deposit number of ATCC No. PTA-4895. Instill another aspect, the invention is an antibody comprising one ormore CDR(s) encoded by (a) a polynucleotide that is produced by a hostcell with a deposit number of ATCC No. PTA-4894; and/or (b) a heavychain that is encoded by a polynucleotide that is produced by a hostcell with a deposit number of ATCC No. PTA-4895.

[0017] In some embodiments, the antibody comprises the human heavy chainIgG2a constant region. In some embodiments the antibody comprises thehuman light chain kappa constant region. In some embodiments, theantibody comprises a modified constant region, such as a constant regionthat is immunologically inert, e.g., does not trigger complementmediated lysis, or does not stimulate antibody-dependent cell mediatedcytotoxicity (ADCC). In other embodiments, the constant region ismodified as described in Eur. J. Immunol. (1999) 29:2613-2624; PCTApplication No. PCT/GB99/01441; and/or UK Patent Application No.9809951.8. In still other embodiments, the antibody comprises a humanheavy chain IgG2a constant region comprising the following mutations:A330P331 to S330S331 (amino acid numbering with reference to thewildtype IgG2a sequence). Eur. J. Immunol. (1999) 29:2613-2624.

[0018] In another aspect, the invention provides polypeptides (which mayor may not be an antibody) comprising any one or more of the following:a) one or more CDR(s) of antibody E3 shown in FIGS. 1A and 1B; b) CDR H3from the heavy chain of antibody E3 shown in FIG. 1A; c) CDR L3 from thelight chain of antibody E3 shown in FIG. 1B; d) three CDRs from thelight chain of antibody E3 shown in FIG. 1B; e) three CDRs from theheavy chain of antibody E3 shown in FIG. 1A; and f) three CDRs from thelight chain and three CDRs from the heavy chain, of antibody E3 shown inFIGS. 1A and 1B. The invention further provides polypeptides (which mayor may not be an antibody) comprising any one or more of the following:a) one or more (one, two, three, four, five, or six) CDR(s) derived fromantibody E3 shown in FIGS. 1A and 1B; b) a CDR derived from CDR H3 fromthe heavy chain of antibody E3 shown in FIG. 1A; and/or c) a CDR derivedfrom CDR L3 from the light chain of antibody E3 shown in FIG. 1B. Insome embodiments, the CDRs may be Kabat CDRs, Chothia CDRs, or acombination of Kabat and Chothia CDRs (termed “extended” or “combined”CDRs herein). In some embodiments, polypeptides (such as an antibody)bind NGF (such as human NGF). In some embodiments, the polypeptidescomprise any of the CDF configurations (including combinations,variants, etc.) described herein.

[0019] In one aspect, the invention provides polypeptides (such as anantibody), which comprise a heavy chain variable region comprising SEQID NO:9, wherein I34 is S, L, V A, or I; and N35 is substituted with N,T or S. For convenience herein, “substituted” or “is” in this context orreference to an amino acid refers to choices of amino acid(s) for agiven position. As is clear, the substitution, or choice, may be theamino acid depicted in a SEQ ID or Figure.

[0020] In another aspect, the invention provides polypeptides (such asan antibody) which comprise a heavy chain variable region comprising SEQID NO:10, wherein M50 is M, I, G, Q, S, or L; A62 is A, or S; and L63 isLor V.

[0021] In another aspect, the invention provides polypeptides (such asan antibody) which comprises a heavy chain variable region comprisingSEQ ID NO: 11, wherein Y100 is Y, L, or R; wherein Y101 is Y or W;wherein G103 is G, A, or S; wherein T104 is T or S; wherein S105 is S,A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; whereinF108 is F or W; wherein D109 is D, N, or G; and wherein Y110 is Y, K, S,R or T.

[0022] In another aspect, the invention provides polypeptides (such asan antibody) which comprise a heavy chain variable region comprising SEQID NO:11, wherein Y100 is Y, L, or R; wherein Y101 is Y or W; whereinG103 is G, A, or S; wherein T104 is T or S; wherein S105 is S, A, or T;wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F108 is For W; wherein D109 is S, A, C, G, D, N, T, or G; and wherein Y110 is anyamino acid.

[0023] In another aspect, the invention provides polypeptides (such asan antibody) which comprise a heavy chain variable region comprising SEQID NO:11, wherein G98 is G, S, A, C, V, N, D, or T; wherein G99 is G, S,A, C, V, N, D, or T; wherein Y100 is Y, L, or R; wherein Y101 is Y or W;wherein G103 is G, A, or S; wherein T104 is T or S; wherein S105 is S,A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; whereinF108 is F or W; wherein D109 is S, A, C, G, D, N, T, or G; and whereinY110 is any amino acid.

[0024] In another aspect, the invention provides polypeptides (such asan antibody) which comprise a light chain variable region comprising SEQID NO:12, wherein S26 is S or F; D28 is D, S, A, or Y; and H32 is H, N,or Q.

[0025] In another aspect, the invention provides polypeptides (such asan antibody) which comprise a light chain variable region comprising SEQID NO: 13, wherein 151 is I, T, V or A; and S56 is S or T.

[0026] In another aspect, the invention provides polypeptides (such asan antibody) which comprise a light chain variable region comprising SEQID NO:14, wherein S91 is S or E; K92 is K, H, R, or S; and wherein Y96is Y or R.

[0027] In another aspect, the invention provides polypeptides (such asan antibody) which comprise a light chain variable region comprising SEQID NO:14, wherein S91 is S or E; K92 is any amino acid; T93 is any aminoacid; and wherein Y96 is Y or R.

[0028] In one aspect, the invention provides polypeptides (such as anantibody), which comprise an amino acid sequence shown in SEQ ID NO:9,wherein I34 is S, L, V A, or I; and N35 is N, T or S.

[0029] In another aspect, the invention provides polypeptides (such asan antibody) which comprise an amino acid sequence shown in SEQ IDNO:10, wherein M50 is M, I, G, Q, S, or L; A62 is A, or S; and L63 is Lor V.

[0030] In another aspect, the invention provides polypeptides (such asan antibody) which comprise an amino acid sequence shown in SEQ IDNO:11, wherein Y100 is Y, L, or R; wherein Y101 is Y or W; wherein G103is G, A, or S; wherein T104 is T or S; wherein S105 is S, A, or T;wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F108 is For W; wherein D109 is D, N, or G; and wherein Y110 is Y, K, S, R or T.

[0031] In another aspect, the invention provides polypeptides (such asan antibody) which comprise an amino acid sequence shown in SEQ IDNO:11, wherein Y100 is Y, L, or R; wherein Y101 is Y or W; wherein G103is G, A, or S; wherein T104 is T or S; wherein S105 is S, A, or T;wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F108 is For W; wherein D 109 is S, A, C, G, D, N, T, or G; and wherein Y110 isany amino acid.

[0032] In another aspect, the invention provides polypeptides (such asan antibody) which comprise an amino acid sequence shown in SEQ IDNO:11, wherein G98 is G, S, A, C, V, N, D, or T; wherein G99 is G, S, A,C, V, N, D, or T; wherein Y100 is Y, L, or R; wherein Y101 is Y or W;wherein G103 is G, A, or S; wherein T104 is T or S; wherein S105 is S,A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; whereinF108 is F or W; wherein D109 is S, A, C, G, D, N, T, or G; and whereinY110 is any amino acid.

[0033] In another aspect, the invention provides polypeptides (such asan antibody) which comprise an amino acid sequence shown in SEQ IDNO:12, wherein S26 is S or F; D28 is D, S, A, or Y; and H32 is H, N, orQ.

[0034] In another aspect, the invention provides polypeptides (such asan antibody) which comprise an amino acid sequence shown in SEQ IDNO:13, wherein 151 is I, T, V or A; and S56 is S or T.

[0035] In another aspect, the invention provides polypeptides (such asan antibody) which comprise an amino acid sequence shown in SEQ IDNO:14, wherein S91 is S or E; K92 is K, H, R, or S; and wherein Y96 is Yor R.

[0036] In another aspect, the invention provides polypeptides (such asan antibody) which comprise an amino acid sequence shown in SEQ IDNO:14, wherein S91 is S or E; K92 is any amino acid; T93 is any aminoacid; and wherein Y96 is Y or R.

[0037] In another aspect, the invention provides polypeptides (such anantibodies, including humanized antibodies) which comprise a heavy chainvariable region comprising the CDR1 region of SEQ ID NO:9, wherein I34is S, L, V A, or I; and N35 is N, T or S; the CDR2 region of SEQ IDNO:10, wherein M50 is M, I, G, Q, S, or L; A62 is A, or S; and L63 is Lor V; and the CDR3 region of SEQ ID NO:11, wherein Y100 is Y, L, or R;wherein Y101 is Y or W; wherein G103 is G, A, or S; wherein T104 is T orS; wherein S105 is S, A, or T; wherein Y106 is Y, R, T, or M; whereinY107 is Y or F; wherein F108 is F or W; wherein D109 is D, N, or G;wherein Y110 is Y, K, S, R or T. In some embodiments, the heavy chainvariable region comprises the CDR3 region of SEQ ID NO:11, wherein Y100is Y, L, or R; wherein Y101 is Y or W; wherein G103 is G, A, or S;wherein T104 is T or S; wherein S105 is S, A, or T; wherein Y106 is Y,R, T, or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D109is S, A, C, G, D, N, T, or G; wherein Y110 is any amino acid. In otherembodiments, the heavy chain variable region comprises the CDR3 regionof SEQ ID NO:11, wherein G98 is G, S, A, C, V, N, D, or T; wherein G99is G, S, A, C, V, N, D, or T; wherein Y100 is Y, L, or R; wherein Y101is Y or W; wherein G103 is G, A, or S; wherein T104 is T or S; whereinS105 is S, A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y orF; wherein F108 is F or W; wherein D109 is S, A, C, G, D, N, T, or G;and wherein Y110 is any amino acid. In some embodiments, the polypeptide(such as an antibody) further comprises an antibody light chain variableregion.

[0038] In another aspect, the invention provides polypeptides (such asan antibody) which comprise a light chain variable region comprising theCDR1 region of SEQ ID NO:12, wherein S26 is S or F; D28 is D, S, A, orY; and H32 is H, N, or Q; the CDR2 region of SEQ ID NO:13, wherein 151is I, T, V or A; and S56 is S or T; and the CDR3 region of SEQ ID NO:14,wherein S91 is S or E; K92 is K, H, R, or S; and wherein Y96 is Y or R.In some embodiments, the light chain variable region comprises the CDR3region of SEQ ID NO:14, wherein S91 is S or E; K92 is any amino acid;T93 is any amino acid; and wherein Y96 is Y or R. In some embodiments,the polypeptide (such as an antibody) further comprises an antibodyheavy chain.

[0039] In another aspect, the invention provides polypeptides (such asan antibody) which comprise (a) a heavy chain variable region comprisingthe CDR1 region of SEQ ID NO:9, wherein I34 is S, L, V A, or I; and N35is N, T or S; the CDR2 region of SEQ ID NO:10, wherein M50 is M, I, G,Q, S, or L; A62 is A, or S; and L63 is L or V; and the CDR3 region ofSEQ ID NO:11, wherein Y100 is Y, L, or R; wherein Y10⁻¹ is Y or W;wherein G103 is G, A, or S; wherein T104 is T or S; wherein S105 is S,A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; whereinF108 is F or W; wherein D109 is D, N, or G; wherein Y110 is Y, K, S, Ror T; and (b) a light chain variable region comprising the CDR1 regionof SEQ ID NO:12, wherein S26 is S or F; D28 is D, S, A, or Y; and H32 isH, N, or Q; the CDR2 region of SEQ ID NO:13, wherein 151 is I, T, V orA; and S56 is S or T; and the CDR3 region of SEQ ID NO:14, wherein S91is S or E; K92 is K, H, R, or S; and wherein Y96 is Y or R. In someembodiments, the light chain variable region comprises the CDR3 regionof SEQ ID NO:14, wherein S91 is S or E; K92 is any amino acid; T93 isany amino acid; and wherein Y96 is Y or R. In some embodiments, theheavy chain variable region comprises the CDR3 region of SEQ ID NO:11,wherein Y100 is Y, L, or R; wherein Y101 is Y or W; wherein G103 is G,A, or S; wherein T104 is T or S; wherein S105 is S, A, or T; whereinY106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F108 is F or W;wherein D109 is S, A, C, G, D, N, T, or G; wherein Y110 is any aminoacid. In other embodiments, the heavy chain variable region comprisesthe CDR3 region of SEQ ID NO:11, wherein G98 is G, S, A, C, V, N, D, orT; wherein G99 is G, S, A, C, V, N, D, or T; wherein Y100 is Y, L, or R;wherein Y101 is Y or W; wherein G103 is G, A, or S; wherein T104 is T orS; wherein S105 is S, A, or T; wherein Y106 is Y, R, T, or M; whereinY107 is Y or F; wherein F108 is F or W; wherein D109 is S, A, C, G, D,N, T, or G; and wherein Y110 is any amino acid. In some embodiments, thepolypeptide further comprises an antibody light chain.

[0040] In another aspect, the invention provides polypeptides (such anantibody, including a humanized antibody) which comprise an amino acidsequence shown in SEQ ID NO:9, wherein I34 is S, L, V A, or I; and N35is N, T or S; an amino acid sequence shown in SEQ ID NO:10, wherein M50is M, I, G, Q, S, or L; A62 is A, or S; and L63 is L or V; and an aminoacid sequence shown in SEQ ID NO:11, wherein Y100 is Y, L, or R; whereinY101 is Y or W; wherein G103 is G, A, or S; wherein T104 is T or S;wherein S105 is S, A, or T; wherein Y106 is Y, R, T, or M; wherein Y107is Y or F; wherein F108 is F or W; wherein D109 is D, N, or G; whereinY110 is Y, K, S, R or T. In some embodiments, the polypeptide comprisesan amino acid sequence shown in SEQ ID NO:11, wherein Y100 is Y, L, orR; and wherein Y101 is Y or W; wherein G103 is G, A, or S; wherein T104is T or S; wherein S105 is S, A, or T; wherein Y106 is Y, R, T, or M;wherein Y107 is Y or F; wherein F108 is F or W; wherein D109 is S, A, C,G, D, N, T, or G; and wherein Y110 is any amino acid. In otherembodiments, the polypeptide comprises an amino acid sequence shown inSEQ ID NO:11, wherein G98 is G, S, A, C, V, N, D, or T; wherein G99 isG, S, A, C, V, N, D, or T; wherein Y100 is Y, L, or R; wherein Y101 is Yor W; wherein G103 is G, A, or S; wherein T104 is T or S; wherein S105is S, A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F;wherein F108 is F or W; wherein D109 is S, A, C, G, D, N, T, or G; andwherein Y110 is any amino acid. In some embodiments, the polypeptide(such as an antibody) further comprises an antibody light chain variableregion.

[0041] In another aspect, the invention provides polypeptides (such asan antibody) which comprise an amino acid sequence shown in SEQ IDNO:12, wherein S26 is S or F;

[0042] D28 is D, S, A, or Y; and H32 is H, N, or Q; an amino acidsequence shown in SEQ ID NO:13, wherein 151 is I, T, V or A; and S56 isS or T; and an amino acid sequence shown in SEQ ID NO:14, wherein S91 isS or E; K92 is K, H, R, or S; and wherein Y96 is Y or R. In someembodiments, the polypeptide comprises an amino acid sequence shown inSEQ ID NO:14, wherein S91 is S or E; K92 is any amino acid; T93 is anyamino acid; and wherein Y96 is Y or R. In some embodiments, thepolypeptide (such as an antibody) further comprises an antibody heavychain variable region.

[0043] In another aspect, the invention provides polypeptides (such asan antibody) which comprise (a) an amino acid sequence shown in SEQ IDNO:9, wherein I34 is S, L, V A, or I; and N35 is N, T or S; an aminoacid sequence shown in SEQ ID NO:10, wherein M50 is M, I, G, Q, S, or L;A62 is A, or S; and L63 is L or V; and an amino acid sequence shown inSEQ ID NO:11, wherein Y100 is Y, L, or R; wherein Y101 is Y or W;wherein G103 is G, A, or S; wherein T104 is T or S; wherein S105 is S,A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; whereinF108 is F or W; wherein D109 is D, N, or G; and wherein Y110 is Y, K, S,R or T; and (b) an amino acid sequence shown in SEQ ID NO:12, whereinS26 is S or F; D28 is D, S, A, or Y; and H32 is H, N, or Q; an aminoacid sequence shown in SEQ ID NO:13, wherein 151 is I, T, V or A; andS56 is S or T; and an amino acid sequence shown in SEQ ID NO:14, whereinS91 is S or E; K92 is K, H, R, or S; and wherein Y96 is Y or R. In someembodiments, the polypeptide comprises an amino acid sequence shown inSEQ ID NO:14, wherein S91 is S or E; K92 is any amino acid; T93 is anyamino acid; and wherein Y96 is Y or R. In some embodiments, thepolypeptide comprises an amino acid sequence shown in SEQ ID NO:11,wherein Y100 is Y, L, or R; wherein Y101 is Y or W; wherein G 103 is G,A, or S; wherein T104 is T or S; wherein S105 is S, A, or T; whereinY106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F108 is F or W;wherein D109 is S, A, C, G, D, N, T, or G; wherein Y110 is any aminoacid. In other embodiments, the polypeptide comprises an amino acidsequence shown in SEQ ID NO:11, wherein G98 is G, S, A, C, V, N, D, orT; wherein G99 is G, S, A, C, V, N, D, or T; wherein Y100 is Y, L, or R;wherein Y101 is Y or W; wherein G 103 is G, A, or S; wherein T104 is Tor S; wherein S105 is S, A, or T; wherein Y106 is Y, R, T, or M; whereinY107 is Y or F; wherein F108 is F or W; wherein D109 is S, A, C, G, D,N, T, or G; and wherein Y110 is any amino acid. In some embodiments, thepolypeptide further comprises an antibody light chain variable region.

[0044] In another aspect, the invention provides polypeptide (such asantibodies) comprising a heavy chain variable region comprising: (a) aCDR1 region of SEQ ID NO:9, wherein I34 is S, L, V A, or I; and N35 issubstituted with N, T or S; (b) a CDR2 region of SEQ ID NO:10, whereinM50 is I, G, Q, S, or L; A62 is A, or S; and L63 is L or V; and (c) aCDR3 region of SEQ ID NO:11, wherein Y100 is Y, L, or R; wherein Y101 isY or W; wherein G103 is G, A, or S; wherein T104 is T or S; wherein S105is S, A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F;wherein F108 is F or W; wherein D109 is D, N, or G; and wherein Y110 isY, K, S, R or T; wherein the antibody binds NGF.

[0045] In another aspect, the invention provides polypeptides (such asantibodies) comprising a light chain variable region comprising: (a) aCDR1 region of SEQ ID NO:12, wherein S26 is S or F; D28 is D, S, A, orY; and H32 is H, N, or Q; (b) a CDR2 region of SEQ ID NO:13, wherein 151is I, T, V or A; and S56 is S or T; and (c) a CDR3 region of SEQ IDNO:14, wherein K92 is K, H, R, or S; and wherein Y96 is Y or R; whereinthe antibody binds NGF.

[0046] In another aspect, the invention provides polypeptides (such asantibodies) comprising (a) a heavy chain variable region comprising: (i)a CDR1 region of SEQ ID NO:9, wherein I34 is substituted with S, L, V A,or I; and N35 is substituted with N, T or S; (ii) a CDR2 region of SEQID NO:10, wherein M50 is I, G, Q, S, or L; A62 is A, or S; and L63 is Lor V; and (iii) a CDR3 region of SEQ ID NO:11, wherein Y100 is Y, L, orR; wherein Y101 is Y or W; wherein G103 is G, A, or S; wherein T104 is Tor S; wherein S105 is S, A, or T; wherein Y106 is Y, R, T, or M; whereinY107 is Y or F; wherein F108 is F or W; wherein D109 is D, N, or G;wherein Y110 is Y, K, S, R or T; and (b) a light chain variable regioncomprising: (i) a CDR1 region of SEQ ID NO:12, wherein S26 is S or F;D28 is D, S, A, or Y; and H32 is H, N, or Q; (ii) a CDR2 region of SEQID NO:13, wherein 151 is I, T, V or A; and S56 is S or T; and (iii) aCDR3 region of SEQ ID NO:14, wherein S91 is S or E; K92 is K, H, R, orS; and wherein Y96 is Y or R; wherein the antibody binds NGF.

[0047] Unless otherwise noted, choice (e.g., substitution) of an aminoacid in one location is independently selected from selection of anamino acid in any other location.

[0048] In some embodiments, polynucleotides (such as an antibody) bindNGF (such as human NGF). In some embodiments, the polypeptides compriseany of the CDR configurations (including combinations, variations, etc.)described herein.

[0049] As is evident from the description herein, the variable regionnumbering used herein is sequential numbering. One of skill in the artreadily understands that a number of antibody numbering systems exist(such as Kabat and Chothia numbering), and how to convert sequentialnumbering into another numbering system, such as Kabat numbering orChothia numbering.

[0050] In another aspect, the invention provides a polypeptide (such asan antibody) comprising an amino acid sequence (such as a CDR3 sequence)selected from SEQ ID NO:46 or 50. In still other embodiments, thepolypeptide further comprises one or more of the amino acid sequencesshown in SEQ ID NOS:3, 4, 5, 6, 7, and 8. In still other embodiments,the polypeptide further comprises one of more of the amino acidsequences shown in SEQ ID NOS:9, 10, 11, 12, 13, 14, and 15.

[0051] In another aspect, the invention provides a polypeptide (such asan antibody) comprising an amino acid sequence (such as a CDR region,such as a CDRH1 and/or CDR H2 region) selected from (a) SEQ ID NOS:28and/or 29; (b) SEQ ID NOS:30 and/or 31; (c) SEQ ID NOS:32 and/or 33; (d)SEQ ID NOS:34 and/or 35; (e) SEQ ID NOS:36 and/or 37; (f) SEQ ID NOS:38and/or 39; and (g) SEQ ID NOS:40 and 41. In some embodiments, thepolypeptide comprises an amino acid sequence (such as a CDR HI region)selected from SEQ ID NOS:28, 30, 32, 34, 36, 38, and 40. In someembodiments, the polypeptide comprises an amino acid sequence (such as aCDR H2 region) selected from SEQ ID NOS:29, 31, 33, 35, 37, 39 and 41.In still other embodiments, the polypeptide further comprises one ormore of the amino acid sequences shown in SEQ ID NOS:3, 4, 5, 6, 7, and8. In still other embodiments, the polypeptide further comprises one ofmore of the amino acid sequences shown in SEQ ID NOS:9, 10, 11, 12, 13,14, and 15.

[0052] In another aspect, the invention provides a polypeptide (such asan antibody) comprising an amino acid sequence (such as a CDR region,such as a CDRL1 and/or CDR L2 region) selected from (a) SEQ ID NOS:18and/or 19; (b) SEQ ID NOS:20 and/or 21; and (c) SEQ ID NOS:22 and/or 23.In some embodiments, the polypeptide comprises an amino acid sequence(such as a CDR L1 region) selected from SEQ ID NOS:18, 20, and 22. Insome embodiments, the polypeptide comprises an amino acid sequence (suchas a CDR L2 region) selected from SEQ ID NOS:19, 21, and 23. In stillother embodiments, the polypeptide further comprises one or more of theamino acid sequences shown in SEQ ID NOS:3, 4, 5, 6, 7, 8. In stillother embodiments, the polypeptide further comprises one of more of theamino acid sequences shown in SEQ ID NOS:9, 10, 11, 12, 13, 14, and 15.

[0053] In another aspect, the invention provides a polypeptide (such asan antibody) comprising an amino acid sequence (such as a CDR region,such as a CDRL3 and/or CDR H3 region) selected from (a) SEQ ID NOS:51and/or 52; (b) SEQ ID NOS:55 and/or 56; (c) SEQ ID NOS:57 and/or 58; (c)SEQ ID NOS:59 and/or 60; (d) SEQ ID NOS:61 and/or 62; (e) SEQ ID NOS:63and/or 64. In some embodiments, the polypeptide comprises an amino acidsequence (such as a CDR L3 region) selected from SEQ ID NOS:51, 55, 57,59, 61, and 63. In some embodiments, the polypeptide comprises an aminoacid sequence (such as a CDR H3 region) selected from SEQ ID NOS:52, 56,58, 60, 62, and 64. In still other embodiments, the polypeptide furthercomprises an amino acid sequence shown in one or more of SEQ ID NOS:18,19, 30 and 31. In still other embodiments, the polypeptide furthercomprises one or more of the amino acid sequences shown in SEQ ID NOS:3,4, 5, 6, 7, and 8. In still other embodiments, the polypeptide furthercomprises one of more of the amino acid sequences shown in SEQ ID NOS:9,10, 11, 12, 13, 14, and 15.

[0054] In another aspect, the invention provides a polypeptide (such asan antibody) comprising one or more of an amino acid sequence (such as aCDR region) shown in SEQ ID NOS:61, 63, 18, 19, 30 and 31.

[0055] In one aspect, the invention provides an anti-NGF antibody (suchas an antagonist antibody) that binds NGF (such as human NGF) with ahigh affinity. In some embodiments, high affinity is (a) binding NGFwith a K_(D) of less than about 2 nM (such as any of about 1 pM, 800 pM,600 pM, 400 pM, 200 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, orless), and/or a k_(off) of slower than about 6×10⁻⁵ s-1); and/or (b)inhibiting (reducing, and/or blocking) human NGF-dependent survival ofmouse E13.5 trigeminal neurons with an IC50 (in the presence of about 15pM of NGF) of about any of 200 pM, 150 pM, 100 pM, 80 pM, 60 pM, 40 pM,20 pM, 10 pM, or less; and/or (c) inhibiting (reducing, and/or blocking)human NGF-dependent survival of mouse E13.5 trigeminal neurons with anIC50 (in the presence of about 1.5 pM of NGF) of about any of 50 pM, 40pM, 30 pM, 10 pM, 20 pM, 10 pM, 5 pM, 2 pM, 1 pM, or less; and/or (d)inhibiting (reducing, and/or blocking) rat NGF-dependent survival ofmouse E13.5 trigeminal neurons with an IC50 (in the presence of about 15pM of NGF) of about any of 150 pM, 125 pM, 100 pM, 80 pM, 60 pM, 40 pM,30 pM, 20 pM, 10 pM, 5 pM, or less; and/or (e) inhibiting (reducing,and/or blocking) rat NGF-dependent survival of mouse E13.5 trigeminalneurons with an IC50 (in the presence of about 1.5 pM of NGF) of aboutany of 30 pM, 25 pM, 20 pM, 15 pM, 10 pM, 5 pM, 4 pM, 3 pM, 2 pM, 1 pM,or less; and/or (f) and/or bind NGF with higher affinity than does thetrkA receptor.

[0056] In another aspect, the invention provides polypeptides (such asan antibody), wherein the polypeptides (a) bind NGF (such as human NGF)with a K_(D) of less than about 2 nM (such as any of about 1 nM, 800 pM,600 pM, 400 pM, 200 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, orless), and/or a k_(off) of slower than about 6×10⁻⁵ s-1); and/or (b)inhibit human NGF-dependent survival of mouse E13.5 trigeminal neuronswith an IC50 (in the presence of about 15 pM of NGF) of about any of 200pM, 150 pM, 100 pM, 80 pM, 60 pM, 40 pM, 20 pM, 10 pM, or less; and/or(c) inhibit human NGF-dependent survival of mouse E13.5 trigeminalneurons with an IC50 (in the presence of about 1.5 pM of NGF) of aboutany of 50 pM, 40 pM, 30 pM, 10 pM, 20 pM, 10 pM, 5 pM, 2 pM, 1 pM, orless; and/or bind NGF with higher affinity than does the trkA receptor.In some embodiments, the polypeptides (a) bind NGF with a K_(D) of lessthan about 2 nM; and/or (b) inhibit human NGF-dependent survival ofmouse E13.5 trigeminal neurons with an IC50 of about 100 pM or less,wherein the IC50 is measured in the presence of about 15 pM NGF; and/or(c) inhibit human NGF-dependent survival of mouse E13.5 trigeminalneurons with an IC50 of about 10 pM or less, wherein the IC50 ismeasured in the presence of about 1.5 pM of NGF, wherein the IC50 ismeasured in the presence of about 15 pM NGF. In some embodiments, thepolypeptides (a) bind NGF with a K_(D) of less than about 100 pM; and/or(b) inhibit human NGF-dependent survival of mouse E13.5 trigeminalneurons with an IC50 of about 20 pM or less, wherein the IC50 ismeasured in the presence of about 15 pM NGF; and/or (c) inhibit humanNGF-dependent survival of mouse E13.5 trigeminal neurons with an IC50 ofabout 2 pM or less, wherein the IC50 is measured in the presence ofabout 1.5 pM of NGF.

[0057] As is evident from the description herein, specifically excludedfrom the invention are polypeptide embodiments consisting of theidentical amino acid sequence to an amino acid sequence of mousemonoclonal antibody, 911. The extended CDR sequences of Mab 911 areshown in FIGS. 1A and 1B, and in SEQ ID NOS:9-14.

[0058] In some embodiments, the invention provides any of the abovepolypeptides or antibodies, further wherein the polypeptide (such as anantibody) is isolated. In some embodiments, the polypeptide (such as anantibody) is substantially purified. In still other embodiments, thepolypeptide (such as an antibody) is affinity matured. In otherembodiments, the antibody is an antagonist antibody. In someembodiments, the polypeptide (such as an antibody) comprises humanframework sequences. In still other embodiments, the polypeptide (suchas an antibody) comprises one or more non-human framework residues. Insome embodiments, the polypeptide (such as an antibody) binds NGF (suchas human NGF) with a K_(D) of 2 nM or less. In some embodiments, thepolypeptide comprises one or more (such as 2, 3, 4, 5, 6, 7, 8, or more)human amino acid substitutions relative to a non-human amino acidsequence (such as a variable region sequence, such as a CDR sequence,such as a framework sequence). In some embodiments, the polypeptidecomprises at least 1, at least 2, or more such as at least 3, 4, 5, 6,or more amino acid substitutions relative to a parent polypeptide aminoacid sequence (such as an antibody 911 amino acid sequence, such as anyone or more of SED ID NOs 9-14). In some embodiments, the bindingaffinity of the antibody has been altered (in some embodiments,increased) relative to a parent antibody (such as Mab 911) affinity. Instill other embodiments, the binding affinity of the antibody is lowerthan the binding affinity of trkA receptor for NGF (such as human NGF).In some embodiments, the polypeptides may be antibodies. In someembodiments, the antibodies are human antibodies. In other embodiments,the antibodies are humanized antibodies. In still other embodiments, theantibodies are monoclonal antibodies. In some embodiments, the antibodyis an affinity matured antibody.

[0059] The invention provides polynucleotides (including isolatedpolynucleotide) comprising polynucleotides encoding any of theembodiments above.

[0060] In another aspect, the invention provides an isolatedpolynucleotide comprising a polynucleotide encoding a fragment or aregion of the antibody E3 (interchangeably termed “E3” herein). In oneembodiment, the fragment is a light chain of the antibody E3 as shown inFIG. 1B. In another embodiment, the fragment is a heavy chain of theantibody E3 as shown in FIG. 1A. In yet another embodiment, the fragmentcontains one or more variable regions from a light chain and/or a heavychain of the antibody E3. In yet another embodiment, the fragmentcontains one or more complementarity determining regions (CDRs) from alight chain and/or a heavy chain of the antibody E3 as shown in FIGS. 1Aand 1B.

[0061] In another aspect, the invention is an isolated polynucleotidecomprising a polynucleotide that encodes for antibody E3. In someembodiments, the polynucleotide comprises either or both of thepolynucleotide shown in FIGS. 2 and 3.

[0062] In another aspect, the invention is an isolated polynucleotidethat encodes for an E3 light chain with a deposit number of ATCC No.PTA-4893 or ATCC No. PTA-4894. In another aspect, the invention is anisolated polynucleotide that encodes for an E3 heavy chain with adeposit number of ATCC No. PTA-4895. In yet another aspect, theinvention is an isolated polynucleotide comprising (a) a variable regionencoded in the polynucleotide with a deposit number of ATCC No. PTA-4893or PTA-4894 and (b) a variable region encoded in the polynucleotide witha deposit number of ATCC No. PTA-4895. In another aspect, the inventionis an isolated polynucleotide comprising (a) one or more CDR encoded inthe polynucleotide with a deposit number of ATCC No. PTA-4893 orPTA-4894; and/or (b) one or more CDR encoded in the polynucleotide witha deposit number of ATCC No. PTA-4895.

[0063] In another aspect, the invention provides polynucleotidesencoding any of the antibodies (including antibody fragments) orpolypeptides described herein.

[0064] In another aspect, the invention provides vectors (includingexpression and cloning vectors) and host cells comprising any of thepolynucleotide disclosed herein.

[0065] As is evident from the description herein, specifically includedfrom the invention are polynucleotide embodiments consisting of theidentical polynucleotide sequence to a polynucleotide sequence of mousemonoclonal antibody, 911. The extended CDR sequences of Mab 911 areshown in FIGS. 1A and 1B, and in SEQ ID NOS:9-14.

[0066] In another aspect, the invention is a host cell comprising apolynucleotide encoding E3 light chain and a polynucleotide encoding E3heavy chain, wherein the polynucleotide(s) encoding E3 light chain has adeposit number of ATCC No. PTA-4893 and/or ATCC No. PTA-4894, and thepolynucleotide encoding E3 heavy chain has a deposit number of ATCC No.PTA-4895. In some embodiments, the host cell comprises polynucleotidecomprising (a) a variable region encoded in the polynucleotide with adeposit number of ATCC No. PTA-4893 or PTA-4894 and/or (b) a variableregion encoded in the polynucleotide with a deposit number of ATCC No.PTA-4895. In some embodiments, the host cell comprises a polynucleotideencoding (a) one or more CDR encoded in the polynucleotide with adeposit number of ATCC No. PTA-4893 or PTA-4894; and/or (b) one or moreCDR encoded in the polynucleotide with a deposit number of ATCC No.PTA-4895. In some embodiments, the host cell is a mammalian cell.

[0067] In another aspect, the invention is a complex of NGF bound byantibody E3. In another aspect, the complex is isolated. In anotheraspect, the complex is substantially purified.

[0068] In another aspect, the invention is a complex of NGF bound by anyof the antibodies or polypeptides described herein. In another aspect,the complex is isolated. In another aspect, the complex is substantiallypurified.

[0069] In another aspect, the invention is a pharmaceutical compositioncomprising any of the polypeptides (including antibodies such asantibody E3) or polynucleotides described herein, such as pharmaceuticalcompositions comprising the antibody E3 or an antibody comprising afragment of the antibody E3, and a pharmaceutically acceptableexcipient.

[0070] In another aspect, the invention is a method of generatingantibody E3 comprising preparing a host cell comprising an expressionvector that encodes for antibody E3; culturing the host cell or progenythereof under conditions that allow production of antibody E3; andpurifying the antibody E3. In some embodiments, the expression vectorcomprises one or both of the polynucleotide sequences shown in FIGS. 2and 3.

[0071] In another aspect, the invention is a method of generatingantibody E3 comprising expressing a polynucleotide encoding E3 lightchain and a polynucleotide encoding E3 heavy chain in a suitable cell,wherein the polynucleotide encoding E3 light chain has a deposit numberof ATCC No. PTA-4893 and/or ATCC No. PTA-4894, and the polynucleotideencoding E3 heavy chain has a deposit number of ATCC No. PTA-4895;generally followed by recovering and/or isolating the antibody.

[0072] In another aspect, the invention provides methods of generatingany of the polypeptides (such as antibodies) described herein byexpressing one or more polynucleotides encoding the antibody (which maybe separately expressed as a single light or heavy chain, or both alight and a heavy chain may be expressed from one vector) in a suitablecell, generally followed by recovering and/or isolating the antibody orpolypeptides of interest.

[0073] In another aspect, the invention is a method of antagonizing NGF(such as human NGF) biological activity using any of the polypeptides(including antibodies such as antibody E3) disclosed herein. In oneembodiment, the method comprises contacting human nerve growth factorwith any of the polypeptides (including antibody E3) described herein,whereby NGF activity (such as human nerve growth factor activity) isantagonized, reduced, blocked, or suppressed.

[0074] In another aspect, the invention is a method of detecting NGFusing any of the polypeptides (including antibodies, such as theantibody E3) described herein. The presence of NGF is detected bydetecting a complex between NGF and any of the polypeptides describedherein (such as antibody E3). The term “detection” as used hereinincludes qualitative and/or quantitative detection (measuring levels)with or without reference to a control.

[0075] In another aspect, the invention is a method of treating pain byadministering an effective amount of a composition comprising theantibody E3 or any of the polypeptide (including antibody) orpolynucleotide embodiments described herein. In some embodiments, thepain is post-surgical pain.

[0076] In another aspect, the invention is a method for preventing ortreating rheumatoid arthritis pain in an individual by administering aneffective amount of anti-NGF antagonist antibody to the individual. Ithas been shown in accordance with the invention that an anti-NGFantagonist antibody is capable of inhibiting or blocking the painassociated with rheumatoid arthritis. In some embodiments, the pain isalleviated within about 24 hours after administering the anti-NGFantagonist antibody. In some embodiments, the pain is alleviated withinabout 4 days after administering the anti-NGF antagonist antibody. Insome embodiments, the pain is alleviated before observing or in theabsence of an indication of improvement of the inflammatory condition inthe individual.

[0077] In another aspect, the invention provides methods for reducingincidence of rheumatoid arthritis pain, ameliorating rheumatoidarthritis pain, suppressing rheumatoid arthritis pain, palliatingrheumatoid arthritis pain, and/or delaying the onset, development, orprogression of rheumatoid arthritis pain in an individual, said methodcomprising administering an effective amount of anti-NGF antagonistantibody to the individual.

[0078] In another aspect, the invention is a method for preventing ortreating osteoarthritis pain in an individual by administering aneffective amount of anti-NGF antagonist antibody to the individual.

[0079] In another aspect, the invention provides methods for treatinginflammatory cachexia (weight loss) associated with rheumatoid arthritisin an individual comprising administering an effective amount of ananti-NGF antagonist antibody. In another aspect, the invention providesmethods for reducing incidence of osteoarthritis pain, amelioratingosteoarthritis pain, suppressing osteoarthritis pain, palliatingosteoarthritis pain, and/or delaying the onset, development, orprogression of osteoarthritis pain in an individual, said methodcomprising administering an effective amount of anti-NGF antagonistantibody to the individual.

[0080] In another aspect, the invention provides kits and compositionscomprising any one or more of the compositions described herein. Thesekits, generally in suitable packaging and provided with appropriateinstructions, are useful for any of the methods described herein.

[0081] The invention also provides any of the compositions and kitsdescribed for any use described herein whether in the context of use asmedicament and/or use for manufacture of a medicament.

BRIEF DESCRIPTION OF THE FIGURES

[0082]FIG. 1A: shows the amino acid sequence of the heavy chain variableregion of the E3 antibody (labeled “6” and “5+affinity maturation H3).The Chothia CDRs and Kabat CDRs are depicted by underlined text and boldand italicized text, respectively. FIG. 1A also shows the alignment ofthe following heavy chain variable region amino acid sequences; (2)VH4-59 human germline acceptor sequence (labeled “VH4-59” or “2”); (3)the acceptor sequences grafted with the extended CDRs of the mouseantibody 911 (labeled “CDR grafted” or “3”); (4) the CDR graftedacceptor sequences including the V71K substitution (labeled “3+oneframework mutation” or “4”); (5) the clone containing affinity maturedCDRs H1 and H2 (labeled “5” or “4+affinity maturation H1, H2”); andantibody E3 (as described above).

[0083]FIG. 1B: shows the amino acid sequence of the light chain variableregion of the E3 antibody (labeled “5” or “4+affinity maturation L3).The Chothia CDRs and Kabat CDRs are depicted by underlined text and boldand italicized text, respectively. FIG. 1B also shows the alignment ofthe following light chain variable region amino acid sequences: (2) O8human germline acceptor sequence (labeled O“8” or “2”); (3) the acceptorsequences grafted with the extended CDRs of the mouse antibody 911(labeled “CDR grafted” or “3”); (4) the CDR grafted acceptor sequences(labeled ““3+affinity maturation L1, L2” or “4”); (5) the clonecontaining affinity matured CDRs L1 and L2 (labeled “5” or “4+affinitymaturation L3”); and antibody E3 (as described above).

[0084]FIG. 2: shows a polynucleotide comprising a polynucleotidesequence encoding the heavy chain variable region of antibody E3.

[0085]FIG. 3: shows a polynucleotide comprising a polynucleotidesequence encoding the light chain variable region of antibody E3.

[0086]FIG. 4: is a graph depicting NGF-dependent survival of E13.5neurons in the presence of varying concentration of human and rat NGF.The X axis corresponds to NGF concentration (ng/ml) and the Y axiscorresponds to counted neurons.

[0087]FIG. 5: is a graph comparing the NGF blocking effect of variousFabs in the presence of either 0.04 ng/ml of human NGF (approximately1.5 pM; shown in lower panel) or 0.4 ng/ml human NGF (approximately 15pM; shown in upper panel). Survival of E13.5 mouse trigeminal neurons invarious concentrations of Fab E3; murine 911 Fab; and Fab H19-L129 andFab 8L2-6D5 was assessed. The IC50 (in pM) was calculated for each Fabat each NGF concentration, and is shown in Table 9. Fab E3 stronglyblocked human NGF-dependent trigeminal neuron survival, with an IC50 ofapproximately 21 pM in the presence of 15 pM human NGF, and an IC50 ofapproximately 1.2 pM in the presence of 1.5 pM human NGF. Fabs 3C andH19-L129 also strongly blocked human NGF-dependent trigeminal neuronsurvival. In both panels, the X axis corresponds to antibodyconcentration (nM) and the Y axis corresponds to counted neurons. 1.5 pMof NGF was around the IC50, while 15 pM represented a saturatingconcentration of NGF.

[0088]FIG. 6: is a graph comparing the NGF blocking effect of variousFabs in the presence of either 0.04 ng/ml of rat NGF (approximately 1.5pM; shown in lower panel) or 0.4 ng/ml rat NGF (approximately 15 pM;shown in upper panel). Survival of E13.5 mouse trigeminal neurons invarious concentrations of Fab E3; murine Fab 911; and Fab H19-L129 and8L2-6D5 was assessed as described above. The IC50 (in pM) was calculatedfor each Fab at each NGF concentration, and is shown in Table 9. Fab E3strongly blocked human NGF-dependent trigeminal neuron survival, with anIC50 of approximately 31.6 pM in the presence of 15 pM rat NGF, and anIC50 of approximately 1.3 pM in the presence of 1.5 pM rat NGF. Fabs 3Cand H19-L129 also strongly blocked rat NGF-dependent trigeminal neuronsurvival. 1.5 pM of NGF was around the IC50, while 15 pM represented asaturating concentration of NGF. In both panels, the X axis correspondsto antibody concentration (nM) and the Y axis corresponds to countedneurons.

[0089]FIG. 7: is a graph depicting resting pain assessed 24 hours aftersurgery and showing that treatment with 0.02 mg/kg, 0.1 mg/kg, 0.6mg/kg, or 1 mg/kg of anti-NGF antibody E3 reduced pain. “*” indicates astatistically significant difference (p<0.5) from the negative control.

[0090]FIG. 8: is a graph depicting resting pain assessed 24 hours aftersurgery and showing that treatment with 0.5 mg/kg of anti-NGF antibodyE3 significantly (p<0.005) reduced resting pain when injected two hoursafter surgery.

[0091]FIG. 9: is a graph showing the results of BIAcore analysis of thebinding affinity to human NGF of mouse antibody 911 (Fab). Mouseantibody 911 bound NGF with a KD of 3.7 nM, k_(off) of 8.4×10⁻⁵s-1 andk_(on) of 2.2×10⁴Ms⁻¹.

[0092]FIG. 10: is a graph showing the results of BIAcore analysis of thebinding affinity to human NGF of antibody E3 (Fab) (referred to as “3EFab”). E3 bound human NGF with a K_(D) of approximately 0.07 nM (andwith a k_(on) of about 6.0×10⁵ M⁻¹s⁻¹, and a k_(off) of about 4.2×10⁻⁵s⁻¹).

[0093]FIG. 11: is a graph depicting that antibody E3 blocks theinteraction of NGF with its receptors, trkA and p75, as assessed bypercent binding detected between NGF and trkA (shown in black circles)and NGF and p75 (shown as hollow squares). The X axis corresponds toconcentration of antibody 3E (Fab) and the Y axis corresponds to NGFbinding (percent maximum RU). Increased concentrations of Fab E3 blockedthe interaction of NGF with both p75 and trka, as shown by decreasedsignal (measured in RU). When antibody E3 (Fab) concentration equaledNGF concentration, no NGF binding was observed (as shown by a signal ofzero).

[0094]FIG. 12: is a graph depicting the human NGF blocking ability offull antibody E3 and Fab E3. Survival of E13.5 mouse trigeminal neuronsin the presence of human NGF and various concentrations of Fab E3 andantibody E3 was assessed. The X axis corresponds to NGF binding sites(nM) and the Y axis corresponds to normalized count of trigeminal (TG)neurons. Full antibody E3 and Fab 3E showed similar levels of inhibitionof NGF-dependent survival of trigeminal neurons when the concentrationof whole antibody and Fab were normalized to the number of NGF bindingsites (Fab has one binding site and whole antibody has two bindingsites).

[0095]FIG. 13: is a graph depicting the ability of variousconcentrations (20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, and 0.0 nM) ofantibody E3 (solid triangles; referred to as “3E”), antibody 911 (solidcircles), and a trkA receptor immunoadhesin (shaded squares; referred as“trkA-Fc) to inhibit NGF-dependent survival of E13.5 trigeminal neuronsin the presence of 0.4 ng/ml human NGF (saturating conditions). The Xaxis corresponds to concentration of antibody (nM) and the Yconcentration corresponds to counted neurons. These results demonstratedthat antibody E3 blocked NGF significantly better than either mousemonoclonal anti-NGF antibody 911 or the trkA immunoadhesin.

[0096]FIG. 14: is a graph depicting that anti-NGF antagonist antibody E3(termed “3E in the figure”) or Fab 911 did not inhibit the neuronalsurvival promoted by NT3, NT4/5 and MSP, even at antibody concentrationsas high as 200 nM. The data represented mean percent survival after 48hours in culture (±standard error of mean, n=3 for each data point)relative to the survival observed in the positive control for eachexperiment (100% survival of trigeminal neurons grown in the presence ofsaturating NGF concentration). Various concentrations (20 nM, 2 nM, or0.2 nM) of E3 Fab (termed “3E” in the figure) and mouse antibody 911 Fabwere used in the presence of no added neurotrophin (termed “control”),400 pM NGF (termed “NGF-400 pM), 10 nM NT3 (termed “NT3-10 nM) or 600 pMMSP (termed “MSP-600 pM).

[0097]FIG. 15: is a graph depicting that anti-NGF antagonist antibody E3(Fab or full antibody) (termed “3E in the figure”) or mouse antibody 911(Fab or full antibody) did not inhibit the neuronal survival promoted byNT3, NT4/5 and MSP, even at antibody concentrations as high as 200 nMVarious concentrations (200 nM and 80 nM) of E3 Fab and full antibodyand mouse antibody 911 full antibody and Fab were used in the presenceof no added neurotrophins (termed “no factor”), 400 pM NGF (termed“NGF-400 pM), 10 nM NT3 (termed “NT3-10 nM) or 600 pM MSP (termed“MSP-600 pM).

[0098]FIG. 16: is a graph depicting that anti-NGF antagonist antibody E3or Fab E3 did not inhibit survival of E17 nodose neurons promoted byBDNF, NT4/5 or LIF. Mouse anti-NGF antagonist antibody 911 was alsotested, and similar results were observed. Various concentrations (200nM or 80 nM) of full antibody E3 (termed “3E in the figure”), Fab E3,full antibody 911, or Fab 911 were tested in the presence of no addedneurotrophins (termed “no factors”), 400 pM BDNF (termed “BDNF-400 pM),400 pM NT4/5 (termed “NT4/5-400 pM), or 2.5 nM LIF (termed “LIF-2.5 nM).

[0099]FIG. 17: is a graph depicting that anti-NGF antagonist antibody E3or Fab E3 did not inhibit survival of E17 nodose neurons promoted byBDNF, NT4/5 or LIF. Various concentrations (200 nM, 20 nM, 2 nM) of FabE3 (termed “3E in the figure”), or Fab 911 were tested in the presenceof no added neurotrophins (termed “control”), 400 pM BDNF (termed“BDNF-400 pM), 400 pM NT4/5 (termed “NT4/5-400 pM), or 2.5 nM LIF(termed “LIP-2.5 nM).

[0100]FIG. 18: is a graph demonstrating nociceptive response inarthritic rats (rheumatoid arthritis model) after administration ofanti-NGF antibodies (E3 and 911) on D14 and D19. E3 (1 mg/kg, i.v. onday 14 and day 19), 911 (10 mg/kg, i.v. on day 14 and day 19), or indo(indomethacin 3 mg/kg, p.o. daily over 10 days) were administered toarthritic mice. Vocalization intensity values are expressed in mV asmeans±s.e.m.

[0101]FIG. 19: is a graph demonstrating effects of anti-NGF antibodieson body weight in arthritis in rats (rheumatoid arthritis model) afteradministration of anti-NGF antibodies on D14 and D19. E3 (1 mg/kg, i.v.on day 14 and day 19), 911 (10 mg/kg, i.v. on day 14 and day 19), orindo (indomethacin 3 mg/kg, p.o. daily over 10 days) were administeredto arthritic mice. Body weight values are expressed in grams asmean±s.e.m.

[0102]FIG. 20: is a graph demonstrating nociceptive response inarthritic rats (rheumatoid arthritis model) after administration ofdifferent doses of anti-NGF antibody E3 (0.003 mg/kg, 0.03 mg/kg, 0.3mg/kg, and 5 mg/kg) on D14 and D18. Vocalization intensity values areexpressed in mV as means±s.e.m.

[0103]FIG. 21: is a graph demonstrating effects of anti-NGF antibody E3on percentage of weight on Day 14 (normalized to Day 14) in arthriticrats (rheumatoid arthritis model) after administration of differentdoses of anti-NGF antibody E3 (0.03 mg/kg, 0.3 mg/kg, and 5 mg/kg) on D14 and D 18.

[0104]FIG. 22: is a graph demonstrating effects of anti-NGF antibody: E3on weight loss in arthritic rats (rheumatoid arthritis model) afteradministration of different doses of anti-NGF antibody E3 (0.03 mg/kg,0.3 mg/kg, and 5 mg/kg) on D14 and D18. Body weight values werenormalized to Day 0.

[0105]FIG. 23: depicts the E3 heavy chain variable region amino acidsequence (FIG. 23A) and light chain variable region amino acid sequence(FIG. 23B), as numbered using sequential numbering, Kabat numbering, andChothia numbering.

DETAILED DESCRIPTION OF THE INVENTION

[0106] The invention disclosed herein provides anti-NGF antagonistantibodies that bind NGF (such as human NGF) with high affinity. Theinvention further provides antibodies and polypeptides derived from E3that bind NGF, and methods of making and using these antibodies. In someembodiments, the invention provides a humanized antibody, E3, whichbinds to nerve growth factor (“NGF”), and methods of making and usingthis antibody. The invention also provides E3 polypeptides (includingantibodies) that bind NGF, and polynucleotides encoding E3 antibodyand/or polypeptide.

[0107] The invention disclosed herein also provides methods forpreventing and/or treating rheumatoid arthritis pain in an individual byadministration of a therapeutically effective amount of an anti-NGFantagonist antibody.

[0108] The invention disclosed herein also provides methods forpreventing and/or treating osteoarthritis pain in an individual byadministration of a therapeutically effective amount of an anti-NGFantagonist antibody.

[0109] The invention also provides methods for adjusting the affinity ofan antibody and methods for characterizing a CDR region.

[0110] General Techniques

[0111] The practice of the present invention will employ, unlessotherwise indicated, conventional techniques of molecular biology(including recombinant techniques), microbiology, cell biology,biochemistry and immunology, which are within the skill of the art. Suchtechniques are explained fully in the literature, such as, MolecularCloning: A Laboratory Manual, second edition (Sambrook et al., 1989)Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed.,1984); Methods in Molecular Biology, Humana Press; Cell Biology: ALaboratory Notebook (J. E. Cellis, ed., 1998) Academic Press; AnimalCell Culture (R. I. Freshney, ed., 1987); Introduction to Cell andTissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Celland Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths,and D. G. Newell, eds., 1993-1998) J. Wiley and Sons; Methods inEnzymology (Academic Press, Inc.); Handbook of Experimental Immunology(D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors forMammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); CurrentProtocols in Molecular Biology (F. M. Ausubel et al., eds., 1987); PCR:The Polymerase Chain Reaction, (Mullis et al., eds., 1994); CurrentProtocols in Immunology (J. E. Coligan et al., eds., 1991); ShortProtocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997);Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989);Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean,eds., Oxford University Press, 2000); Using antibodies: a laboratorymanual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press,1999); The Antibodies (M. Zanetti and J. D. Capra, eds., HarwoodAcademic Publishers, 1995); and Cancer: Principles and Practice ofOncology (V. T. DeVita et al., eds., J. B. Lippincott Company, 1993).

[0112] Definitions

[0113] An “antibody” is an immunoglobulin molecule capable of specificbinding to a target, such as a carbohydrate, polynucleotide, lipid,polypeptide, etc., through at least one antigen recognition site,located in the variable region of the immunoglobulin molecule. As usedherein, the term encompasses not only intact polyclonal or monoclonalantibodies, but also fragments thereof (such as Fab, Fab′, F(ab′)₂, Fv),single chain (ScFv), mutants thereof, fusion proteins comprising anantibody portion, and any other modified configuration of theimmunoglobulin molecule that comprises an antigen recognition site. Anantibody includes an antibody of any class, such as IgG, IgA, or IgM (orsub-class thereof), and the antibody need not be of any particularclass. Depending on the antibody amino acid sequence of the constantdomain of its heavy chains, immunoglobulins can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. Theheavy-chain constant domains that correspond to the different classes ofimmunoglobulins are called alpha, delta, epsilon, gamma, and mu,respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known

[0114] “Fv” is an antibody fragment that contains a completeantigen-recognition and -binding site. In a two-chain Fv species, thisregion consists of a dimer of one heavy and one light chain variabledomain in tight, non-covalent association. In a single-chain Fv species,one heavy and one light chain variable domain can be covalently linkedby a flexible peptide linker such that the light and heavy chains canassociate in a dimeric structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding specificity on thesurface of the VH-VL dimer. However, even a single variable domain (orhalf of a Fv comprising only 3 CDRs specific for an antigen) has theability to recognize and bind antigen, although generally at a loweraffinity than the entire binding site.

[0115] The Fab fragment also contains the constant domain of the lightchain and the first constant domain (CH1) of the heavy chain. Fab′fragments differ from Fab fragments by the addition of a few residues atthe carboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge regions.

[0116] A “monoclonal antibody” refers to a homogeneous antibodypopulation wherein the monoclonal antibody is comprised of amino acids(naturally occurring and non-naturally occurring) that are involved inthe selective binding of an antigen. A population of monoclonalantibodies is highly specific, being directed against a single antigenicsite. The term “monoclonal antibody” encompasses not only intactmonoclonal antibodies and full-length monoclonal antibodies, but alsofragments thereof (such as Fab, Fab′, F(ab′)₂, Fv), single chain (ScFv),mutants thereof, fusion proteins comprising an antibody portion, and anyother modified configuration of the immunoglobulin molecule thatcomprises an antigen recognition site of the required specificity andthe ability to bind to an antigen. It is not intended to be limited asregards to the source of the antibody or the manner in which it is made(e.g., by hybridoma, phage selection, recombinant expression, transgenicanimals, etc.).

[0117] As used herein, “human antibody” means an antibody having anamino acid sequence corresponding to that of an antibody produced by ahuman and/or has been made using any of the techniques for making humanantibodies known in the art or disclosed herein. This definition of ahuman antibody includes antibodies comprising at least one human heavychain polypeptide or at least one human light chain polypeptide. Onesuch example is an antibody comprising murine light chain and humanheavy chain polypeptides. Human antibodies can be produced using varioustechniques known in the art. In one embodiment, the human antibody isselected from a phage library, where that phage library expresses humanantibodies (Vaughan et al., 1996, Nature Biotechnology, 14:309-314;Sheets et al., 1998, PNAS, (USA) 95:6157-6162; Hoogenboom and Winter,1991, J. Mol. Biol., 227:381; Marks et al., 1991, J. Mol. Biol.,222:581). Human antibodies can also be made by introducing humanimmunoglobulin loci into transgenic animals, e.g., mice in which theendogenous immunoglobulin genes have been partially or completelyinactivated. This approach is described in U.S. Pat. Nos. 5,545,807;5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016.Alternatively, the human antibody may be prepared by immortalizing humanB lymphocytes that produce an antibody directed against a target antigen(such B lymphocytes may be recovered from an individual or may have beenimmunized in vitro). See, e.g., Cole et al., Monoclonal Antibodies andCancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., 1991, J.Immunol., 147 (1):86-95; and U.S. Pat. No. 5,750,373.

[0118] “Chimeric antibodies” refers to those antibodies wherein oneportion of each of the amino acid sequences of heavy and light chains ishomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular class, while theremaining segment of the chains is homologous to corresponding sequencesin another. Typically, in these chimeric antibodies, the variable regionof both light and heavy chains mimics the variable regions of antibodiesderived from one species of mammals, while the constant portions arehomologous to the sequences in antibodies derived from another. Oneclear advantage to such chimeric forms is that, for example, thevariable regions can conveniently be derived from presently knownsources using readily available hybridomas or B cells from non humanhost organisms in combination with constant regions derived from, forexample, human cell preparations. While the variable region has theadvantage of ease of preparation, and the specificity is not affected byits source, the constant region being human, is less likely to elicit animmune response from a human subject when the antibodies are injectedthan would the constant region from a non-human source. However, thedefinition is not limited to this particular example.

[0119] A “functional Fc region” possesses at least one effector functionof a native sequence Fc region. Exemplary “effector functions” includeC1q binding; complement dependent cytotoxicity (CDC); Fc receptorbinding; antibody-dependent cell-mediated cytotoxicity (ADCC);phagocytosis; down-regulation of cell surface receptors (e.g. B cellreceptor; BCR), etc. Such effector functions generally require the Fcregion to be combined with a binding domain (e.g. an antibody variabledomain) and can be assessed using various assays known in the art forevaluating such antibody effector functions.

[0120] A “native sequence Fc region” comprises an amino acid sequenceidentical to the amino acid sequence of an Fc region found in nature. A“variant Fc region” comprises an amino acid sequence which differs fromthat of a native sequence Fc region by virtue of at least one amino acidmodification, yet retains at least one effector function of the nativesequence Fc region. Preferably, the variant Fc region has at least oneamino acid substitution compared to a native sequence Fc region or tothe Fc region of a parent polypeptide, e.g. from about one to about tenamino acid substitutions, and preferably from about one to about fiveamino acid substitutions in a native sequence Fc region or in the Fcregion of the parent polypeptide. The variant Fc region herein willpreferably possess at least about 80% sequence identity with a nativesequence Fc region and/or with an Fc region of a parent polypeptide, andmost preferably at least about 90% sequence identity therewith, morepreferably at least about 95% sequence identity therewith.

[0121] As used herein “antibody-dependent cell-mediated cytotoxicity”and “ADCC” refer to a cell-mediated reaction in which nonspecificcytotoxic cells that express Fc receptors (FcRs) (e.g. natural killer(NK) cells, neutrophils, and macrophages) recognize bound antibody on atarget cell and subsequently cause lysis of the target cell. ADCCactivity of a molecule of interest can be assessed using an in vitroADCC assay, such as that described in U.S. Pat. No. 5,500,362 or5,821,337. Useful effector cells for such assays include peripheralblood mononuclear cells (PBMC) and NK cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in a animal model such as that disclosed in Clynes etal., 1998, PNAS (USA), 95:652-656.

[0122] As used herein, “Fc receptor” and “FcR” describe a receptor thatbinds to the Fc region of an antibody. The preferred FcR is a nativesequence human FcR. Moreover, a preferred FcR is one which binds an IgGantibody (a gamma receptor) and includes receptors of the FcγRI, FcγII,and FcγRIII subclasses, including allelic variants and alternativelyspliced forms of these receptors. FcγRII receptors include FcγRIIA (an“activating receptor”) and FcγIIB (an “inhibiting receptor”), which havesimilar amino acid sequences that differ primarily in the cytoplasmicdomains thereof. FcRs are reviewed in Ravetch and Kinet, 1991, Ann. Rev.Immunol., 9:457-92; Capel et al., 1994, Immunomethods, 4:25-34; and deHaas et al., 1995, J Lab. Clin. Med., 126:330-41. “FcR” also includesthe neonatal receptor, FcRn, which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., 1976, J. Immunol., 117:587;and Kim et al., 1994, J. Immunol., 24:249).

[0123] “Complement dependent cytotoxicity” and “CDC” refer to the lysingof a target in the presence of complement. The complement activationpathway is initiated by the binding of the first component of thecomplement system (C1q) to a molecule (e.g. an antibody) complexed witha cognate antigen. To assess complement activation, a CDC assay, e.g. asdescribed in Gazzano-Santoro et al., J. Immunol. Methods, 202:163(1996), may be performed.

[0124] As used herein, the terms “E3”, “3E”, and “antibody E3” are usedinterchangeably to refer to an antibody comprising the amino acidsequence of the heavy chain and light chain variable regions shown inFIGS. 1A (SEQ ID NO:1) and 1B (SEQ ID NO:2), respectively. The CDRportions of antibody E3 (including Chothia and Kabat CDRs) arediagrammatically depicted in FIGS. 1A and 1B. FIGS. 2 and 3 showpolynucleotides encoding heavy and light chains, respectively,comprising the heavy and light chain variable regions shown in FIGS. 1Aand 1B, respectively. The generation and characterization of E3 isdescribed in the Examples. Different biological functions are associatedwith E3, including, but not limited to, ability to bind to NGF andinhibit NGF biological activity and/or downstream pathway(s) mediated byNGF signaling; and ability to inhibit NGF-dependent survival of mouseE13.5 trigeminal neurons. As discussed herein, antibodies of theinvention may have any one or more of these characteristics. In someembodiments, the term “E3” refers to immunoglobulin encoded by (a) apolynucleotide encoding E3 light chain that has a deposit number of ATCCNo. PTA-4893 or ATCC No. PTA-4894, and (b) a polynucleotide encoding E3heavy chain that has a deposit number of ATCC No. PTA-4895.

[0125] As used herein, “immunospecific” binding of antibodies refers tothe antigen specific binding interaction that occurs between theantigen-combining site of an antibody and the specific antigenrecognized by that antibody (i.e., the antibody reacts with the proteinin an ELISA or other immunoassay, and does not react detectably withunrelated proteins).

[0126] An epitope that “specifically binds”, or “preferentially binds”(used interchangeably herein) to an antibody or a polypeptide is a termwell understood in the art, and methods to determine such specific orpreferential binding are also well known in the art. A molecule is saidto exhibit “specific binding” or “preferential binding” if it reacts orassociates more frequently, more rapidly, with greater duration and/orwith greater affinity with a particular cell or substance than it doeswith alternative cells or substances. An antibody “specifically binds”or “preferentially binds” to a target if it binds with greater affinity,avidity, more readily, and/or with greater duration than it binds toother substances. For example, an antibody that specifically orpreferentially binds to an NGF epitope is an antibody that binds thisepitope with greater affinity, avidity, more readily, and/or withgreater duration than it binds to other NGF epitopes or non-NGFepitopes. It is also understood by reading this definition that, forexample, an antibody (or moiety or epitope) that specifically orpreferentially binds to a first target may or may not specifically orpreferentially bind to a second target. As such, “specific binding” or“preferential binding” does not necessarily require (although it caninclude) exclusive binding. Generally, but not necessarily, reference tobinding means preferential binding.

[0127] The terms “polypeptide”, “oligopeptide”, “peptide” and “protein”are used interchangeably herein to refer to polymers of amino acids ofany length. The polymer may be linear or branched, it may comprisemodified amino acids, and it may be interrupted by non-amino acids. Theterms also encompass an amino acid polymer that has been modifiednaturally or by intervention; for example, disulfide bond formation,glycosylation, lipidation, acetylation, phosphorylation, or any othermanipulation or modification, such as conjugation with a labelingcomponent. Also included within the definition are, for example,polypeptides containing one or more analogs of an amino acid (including,for example, unnatural amino acids, etc.), as well as othermodifications known in the art. It is understood that, because thepolypeptides of this invention are based upon an antibody, thepolypeptides can occur as single chains or associated chains.

[0128] “Polynucleotide,” or “nucleic acid,” as used interchangeablyherein, refer to polymers of nucleotides of any length, and include DNAand RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides,modified nucleotides or bases, and/or their analogs, or any substratethat can be incorporated into a polymer by DNA or RNA polymerase. Apolynucleotide may comprise modified nucleotides, such as methylatednucleotides and their analogs. If present, modification to thenucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter polymerization, such as by conjugation with a labeling component.Other types of modifications include, for example, “caps”, substitutionof one or more of the naturally occurring nucleotides with an analog,internucleotide modifications such as, for example, those with unchargedlinkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates,cabamates, etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelators (e.g., metals, radioactivemetals, boron, oxidative metals, etc.), those containing alkylators,those with modified linkages (e.g., alpha anomeric nucleic acids, etc.),as well as unmodified forms of the polynucleotide(s). Further, any ofthe hydroxyl groups ordinarily present in the sugars may be replaced,for example, by phosphonate groups, phosphate groups, protected bystandard protecting groups, or activated to prepare additional linkagesto additional nucleotides, or may be conjugated to solid supports. The5′ and 3′ terminal OH can be phosphorylated or substituted with aminesor organic capping groups moieties of from 1 to 20 carbon atoms. Otherhydroxyls may also be derivatized to standard protecting groups.Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including, forexample, 2′O-methyl-, 2′-O-allyl, 2′-fluoro- or 2′-azido-ribose,carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such asarabinose, xyloses or lyxoses, pyranose sugars, furanose sugars,sedoheptuloses, acyclic analogs and abasic nucleoside analogs such asmethyl riboside. One or more phosphodiester linkages may be replaced byalternative linking groups. These alternative linking groups include,but are not limited to, embodiments wherein phosphate is replaced byP(O)S(“thioate”), P(S)S (“dithioate”), “(O)NR₂ (“amidate”), P(O)R,P(O)OR, CO or CH₂ (“formacetal”), in which each R or R′ is independentlyH or substituted or unsubstituted alkyl (1-20 C) optionally containingan ether (—O—) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl oraraldyl. Not all linkages in a polynucleotide need be identical. Thepreceding description applies to all polynucleotides referred to herein,including RNA and DNA.

[0129] A “variable region” of an antibody refers to the variable regionof the antibody light chain or the variable region of the antibody heavychain, either alone or in combination. The variable regions of the heavyand light chain each consist of four framework regions (FR) connected bythree complementarity determining regions (CDRs) also known ashypervariable regions. The CDRs in each chain are held together in closeproximity by the FRs and, with the CDRs from the other chain, contributeto the formation of the antigen-binding site of antibodies. There are atleast two techniques for determining CDRs: (1) an approach based oncross-species sequence variability (i.e., Kabat et al. Sequences ofProteins of Immunological Interest, (5th ed., 1991, National Institutesof Health, Bethesda Md.)); and

[0130] (2) an approach based on crystallographic studies ofantigen-antibody complexes (Chothia et al. (1989) Nature 342:877;Al-lazikani et al (1997) J. Molec. Biol. 273:927-948)). As used herein,a CDR may refer to CDRs defined by either approach or by a combinationof both approaches.

[0131] A “constant region” of an antibody refers to the constant regionof the antibody light chain or the constant region of the antibody heavychain, either alone or in combination.

[0132] As used herein, the term “nerve growth factor” and “NGF” refersto nerve growth factor and variants thereof that retain at least part ofthe biological activity of NGF. As used herein, NGF includes allmammalian species of native sequence NGF, including human, canine,feline, equine, or bovine.

[0133] “NGF receptor” refers to a polypeptide that is bound by oractivated by NGF. NGF receptors include the TrkA receptor and the p75receptor of any mammalian species, including, but are not limited to,human, canine, feline, equine, primate, or bovine.

[0134] As used herein, an “anti-NGF antagonist antibody”(interchangeably termed “anti-NGF antibody”) refers to an antibody whichis able to bind to NGF and inhibit NGF biological activity and/ordownstream pathway(s) mediated by NGF signaling. An anti-NGF antagonistantibody encompasses antibodies that block, antagonize, suppress orreduce (including significantly) NGF biological activity, includingdownstream pathways mediated by NGF signaling, such as receptor bindingand/or elicitation of a cellular response to NGF. For purpose of thepresent invention, it will be explicitly understood that the term“anti-NGF antagonist antibody” encompass all the previously identifiedterms, titles, and functional states and characteristics whereby the NGFitself, an NGF biological activity (including but not limited to itsability to ability to mediate any aspect of post-surgical pain), or theconsequences of the biological activity, are substantially nullified,decreased, or neutralized in any meaningful degree. In some embodiments,an anti-NGF antagonist antibody binds NGF and prevent NGF dimerizationand/or binding to an NGF receptor (such as p75 and/or trkA). In otherembodiments, an anti-NGF antibody binds NGF and prevents trkA receptordimerization and/or trkA autophosphorylation. Examples of anti-NGFantagonist antibodies are provided herein.

[0135] “Biological activity” of NGF generally refers to the ability tobind NGF receptors and/or activate NGF receptor signaling pathways.Without limitation, a biological activity includes any one or more ofthe following: the ability to bind an NGF receptor (such as p75 and/ortrkA); the ability to promote trkA receptor dimerization and/orautophosphorylation; the ability to activate an NGF receptor signalingpathway; the ability to promote cell differentiation, proliferation,survival, growth and other changes in cell physiology, including (in thecase of neurons, including peripheral and central neuron) change inneuronal morphology, synaptogenesis, synaptic function, neurotransmitterand/or neuropeptide release and regeneration following damage; theability to promote survival of mouse E13.5 trigeminal neurons; and theability to mediate pain, including post-surgical pain.

[0136] As used herein, “substantially pure” refers to material which isat least 50% pure (i.e., free from contaminants), more preferably atleast 90% pure, more preferably at least 95% pure, more preferably atleast 98% pure, more preferably at least 99% pure.

[0137] A “host cell” includes an individual cell or cell culture thatcan be or has been a recipient for vector(s) for incorporation ofpolynucleotide inserts. Host cells include progeny of a single hostcell, and the progeny may not necessarily be completely identical (inmorphology or in genomic DNA complement) to the original parent cell dueto natural, accidental, or deliberate mutation. A host cell includescells transfected in vivo with a polynucleotide(s) of this invention.

[0138] As used herein, “treatment” is an approach for obtainingbeneficial or desired clinical results. For purposes of this invention,beneficial or desired clinical results include, but are not limited to,one or more of the following: improvement or alleviation of any aspectof pain, including acute, chronic, inflammatory, neuropathic,post-surgical pain, rheumatoid arthritis pain, or osteoarthritis pain.For purposes of this invention, beneficial or desired clinical resultsinclude, but are not limited to, one or more of the following: includinglessening severity, alleviation of one or more symptoms associated withpain including any aspect of pain (such as shortening duration of pain,reduction of pain sensitivity or sensation).

[0139] An “effective amount” of drug, compound, or pharmaceuticalcomposition is an amount sufficient to effect beneficial or desiredresults including clinical results such as alleviation or reduction inpain sensation. An effective amount can be administered in one or moreadministrations. For purposes of this invention, an effective amount ofdrug, compound, or pharmaceutical composition is an amount sufficient totreat, ameliorate, reduce the intensity of and/or prevent pain,including post-surgical pain, rheumatoid arthritis pain, and/orosteoarthritis pain. In some embodiments, the “effective amount” mayreduce pain at rest (resting pain) or mechanically-induced pain(including pain following movement), or both, and it may be administeredbefore, during or after an incision, cut, tear or injury and/or before,during or after painful stimulus. As is understood in the clinicalcontext, an effective amount of a drug, compound, or pharmaceuticalcomposition may or may not be achieved in conjunction with another drug,compound, or pharmaceutical composition. Thus, an “effective amount” maybe considered in the context of administering one or more therapeuticagents, and a single agent may be considered to be given in an effectiveamount if, in conjunction with one or more other agents, a desirableresult may be or is achieved.

[0140] “Reducing incidence” of pain means any of reducing severity(which can include reducing need for and/or amount of (e.g., exposureto) other drugs and/or therapies generally used for this conditions,including, for example, opiates), duration, and/or frequency (including,for example, delaying or increasing time to post-surgical pain in anindividual). As is understood by those skilled in the art, individualsmay vary in terms of their response to treatment, and, as such, forexample, a “method of reducing incidence of rheumatoid arthritis pain orosteoarthritis pain in an individual” reflects administering theanti-NGF antagonist antibody based on a reasonable expectation that suchadministration may likely cause such a reduction in incidence in thatparticular individual.

[0141] “Ameliorating” a pain or one or more symptoms of a pain (such asrheumatoid arthritis pain or osteoarthritis pain) means a lessening orimprovement of one or more symptoms of a pain as compared to notadministering an anti-NGF antagonist antibody. “Ameliorating” alsoincludes shortening or reduction in duration of a symptom.

[0142] “Palliating” a pain or one or more symptoms of a pain (such asrheumatoid arthritis pain or osteoarthritis pain) means lessening theextent of one or more undesirable clinical manifestations ofpost-surgical pain in an individual or population of individuals treatedwith an anti-NGF antagonist antibody in accordance with the invention.

[0143] As used therein, “delaying” the development of pain means todefer, hinder, slow, retard, stabilize, and/or postpone progression ofpain, such as post-surgical pain, rheumatoid arthritis pain, orosteoarthritis pain. This delay can be of varying lengths of time,depending on the history of the disease and/or individuals beingtreated. As is evident to one skilled in the art, a sufficient orsignificant delay can, in effect, encompass prevention, in that theindividual does not develop pain. A method that “delays” development ofthe symptom is a method that reduces probability of developing thesymptom in a given time frame and/or reduces extent of the symptoms in agiven time frame, when compared to not using the method. Suchcomparisons are typically based on clinical studies, using astatistically significant number of subjects.

[0144] “Pain” as used herein refers to pain of any etiology, includingacute and chronic pain, and any pain with an inflammatory component.Examples of pain include post-surgical pain, post-operative pain(including dental pain), migraine, headache and trigeminal neuralgia,pain associated with burn, wound or kidney stone, pain associated withtrauma (including traumatic head injury), neuropathic pain, painassociated with musculo-skeletal disorders such as rheumatoid arthritis,osteoarthritis, ankylosing spondylitis, sero-negative (non-rheumatoid)arthropathies, non-articular rheumatism and peri-articular disorders,and pain associated with cancer (including “break-through pain” and painassociated with terminal cancer), peripheral neuropathy andpost-herpetic neuralgia. Examples of pain with an inflammatory component(in addition to some of those described above) include rheumatic pain,pain associated with mucositis, and dysmenorrhea.

[0145] “Post-surgical pain” (interchangeably termed “post-incisional” or“post-traumatic pain”) refers to pain arising or resulting from anexternal trauma such as a cut, puncture, incision, tear, or wound intotissue of an individual (including that that arises from all surgicalprocedures, whether invasive or non-invasive). As used herein,post-surgical pain does not include pain that occurs (arises ororiginates) without an external physical trauma. In some embodiments,post-surgical pain is internal or external (including peripheral) pain,and the wound, cut, trauma, tear or incision may occur accidentally (aswith a traumatic wound) or deliberately (as with a surgical incision).As used herein, “pain” includes nociception and the sensation of pain,and pain can be assessed objectively and subjectively, using pain scoresand other methods well-known in the art. Post-surgical pain, as usedherein, includes allodynia (i.e., increased response to a normallynon-noxious stimulus) and hyperalgesia (i.e., increased response to anormally noxious or unpleasant stimulus), which can in turn, be thermalor mechanical (tactile) in nature. In some embodiments, the pain ischaracterized by thermal sensitivity, mechanical sensitivity and/orresting pain. In some embodiments, the post-surgical pain comprisesmechanically-induced pain or resting pain. In other embodiments, thepost-surgical pain comprises resting pain. The pain can be primary orsecondary pain, as is well-known in the art.

[0146] A “biological sample” encompasses a variety of sample typesobtained from an individual and can be used in a diagnostic ormonitoring assay. The definition encompasses blood and other liquidsamples of biological origin, solid tissue samples such as a biopsyspecimen or tissue cultures or cells derived therefrom, and the progenythereof. The definition also includes samples that have been manipulatedin any way after their procurement, such as by treatment with reagents,solubilization, or enrichment for certain components, such as proteinsor polynucleotides, or embedding in a semi-solid or solid matrix forsectioning purposes. The term “biological sample” encompasses a clinicalsample, and also includes cells in culture, cell supernatants, celllysates, serum, plasma, biological fluid, and tissue samples.

[0147] An “individual” is a vertebrate, preferably a mammal, morepreferably a human. Mammals include, but are not limited to, farmanimals (such as cows), sport animals, pets (such as cats, dogs andhorses), primates, mice and rats.

[0148] As used herein, “vector” means a construct, which is capable ofdelivering, and preferably expressing, one or more gene(s) orsequence(s) of interest in a host cell. Examples of vectors include, butare not limited to, viral vectors, naked DNA or RNA expression vectors,plasmid, cosmid or phage vectors, DNA or RNA expression vectorsassociated with cationic condensing agents, DNA or RNA expressionvectors encapsulated in liposomes, and certain eukaryotic cells, such asproducer cells.

[0149] As used herein, “expression control sequence” means a nucleicacid sequence that directs transcription of a nucleic acid. Anexpression control sequence can be a promoter, such as a constitutive oran inducible promoter, or an enhancer. The expression control sequenceis operably linked to the nucleic acid sequence to be transcribed.

[0150] As used herein, “pharmaceutically acceptable carrier” includesany material which, when combined with an active ingredient, allows theingredient to retain biological activity and is non-reactive with thesubject's immune system. Examples include, but are not limited to, anyof the standard pharmaceutical carriers such as a phosphate bufferedsaline solution, water, emulsions such as oil/water emulsion, andvarious types of wetting agents. Preferred diluents for aerosol orparenteral administration are phosphate buffered saline or normal (0.9%)saline. Compositions comprising such carriers are formulated by wellknown conventional methods (see, for example, Remington's PharmaceuticalSciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton,Pa., 1990; and Remington, The Science and Practice of Pharmacy 20th Ed.Mack Publishing, 2000).

[0151] The term “K_(off)”, as used herein, is intended to refer to theoff rate constant for dissociation of an antibody from theantibody/antigen complex.

[0152] The term “K_(d)”, as used herein, is intended to refer to thedissociation constant of an antibody-antigen interaction.

[0153] Antibody E3, E3-Derived Antibodies, Compositions, and Methods ofUse

[0154] E3 Compositions, E3 Derived Compositions, and Methods of Makingthe Compositions

[0155] This invention encompasses compositions, including pharmaceuticalcompositions, comprising an E3 antibody or polypeptide; andpolynucleotides comprising sequences encoding an E3 antibody orpolypeptide. As used herein, compositions comprise one or moreantibodies or polypeptides (which may or may not be an antibody) thatbind to NGF, and/or one or more polynucleotides comprising sequencesencoding one or more antibodies or polypeptides that bind to NGF. Thesecompositions may further comprise suitable excipients, such aspharmaceutically acceptable excipients including buffers, which are wellknown in the art.

[0156] The invention also encompasses isolated antibody, polypeptide andpolynucleotide embodiments. The invention also encompasses substantiallypure antibody, polypeptide and polynucleotide embodiments.

[0157] The antibodies and polypeptides of the invention arecharacterized by any (one or more) of the following characteristics: (a)ability to bind to NGF; (b) ability to reduce and/or inhibit NGFbiological activity and/or downstream pathway(s) mediated by NGFsignaling; (c) ability to reduce and/or inhibit NGF-dependent survivalof mouse E13.5 trigeminal neurons; (d) absence of any significantcross-reactivity to NT3, NT4/5, and/or BDNF; (e) ability to treat and/orprevent pain (including post-surgical pain); (f) ability to increaseclearance of NGF; (g) ability to reduce or inhibit activation of trkareceptor, as detected, for example, using kinase receptor activationassay (KIRA) (see U.S. Pat. No. 6,027,927).

[0158] The binding properties of antibody E3, which binds human NGF withhigh affinity and slow dissociation kinetics, compared with parentmurine anti-NGF monoclonal antibody 911, are summarized below. E3 bindshuman NGF with an approximately 50-fold higher binding affinity thanparent mouse antibody 911. antibody k_(D) K_(off) K_(on) 911 (Fab) 3.7nM 9 × 10⁻⁵s⁻¹ 2.2 × 10⁴M⁻¹s⁻¹ E3 (Fab) 0.07 nM <4 × 10⁻⁵s⁻¹     6 ×10⁵M⁻¹s⁻¹

[0159] The E3 antibody and related antibodies also exhibit a strongcapacity to antagonize human NGF, as assessed by in vitro assays (seeExamples 2 and 3). For example, antibody E3 antagonizes theNGF-dependent survival of mouse E13 trigeminal neurons at an IC50 ofabout 21 pM in the presence of 15 pM of human NGF, and about 1.2 pM inthe presence of 1.5 pM of human NGF.

[0160] Accordingly, in another aspect, the antibodies and polypeptidesof the invention are further identified and characterized by: (h) highaffinity binding to human NGF with low dissociation kinetics (in someembodiments, with a K_(D) of less than about 2 nM, and/or a k_(off) ofslower than about 6×10-5 s-1) and/or (i) ability to inhibit (block)NGF-dependent survival of mouse E13.5 trigeminal neurons with an IC50 ofabout 100 pM or less at about 15 pM of NGF (in some embodiments, humanNGF) and/or an IC50 of about 20 pM or less at about 1.5 pM of NGF.

[0161] In some embodiments, the antibody binds human NGF, and does notsignificantly bind an NGF from another vertebrate species (in someembodiment, mammalian). In some embodiments, the antibody binds humanNGF as well as one or more NGF from another vertebrate species (in someembodiments, mammalian). In still other embodiments, the antibody bindsNGF and does not significantly cross-react with other neurotrophins(such as the related neurotrophins, NT3, NT4/5, and/or BDNF). In someembodiments, the antibody binds NGF as well as at least one otherneurotrophin. In some embodiments, the antibody binds to a mammalianspecies of NGF, such as horse or dog, but does not significantly bind toNGF from anther mammalian species.

[0162] In some embodiments, the invention is an antibody comprising alight chain that is encoded by a polynucleotide that is produced by ahost cell with a deposit number of ATCC No. PTA-4893 or ATCC No.PTA-4894. In another aspect, the invention is an antibody comprising aheavy chain that is encoded by a polynucleotide that is produced by ahost cell with a deposit number of ATCC No. PTA-4895. The presentinvention also encompasses various formulations of E3 and equivalentantibody fragments (e.g., Fab, Fab′, F(ab′)₂, Fv, Fc, etc.), singlechain (ScFv), mutants thereof, fusion proteins comprising an antibodyportion, and any other modified configuration of E3 that comprises anantigen (NGF) recognition site of the required specificity. Theequivalent antibodies of E3, including antibody and polypeptidefragments (which may or may not be antibodies) of E3, and polypeptidescomprising polypeptide fragments of E3 are identified and characterizedby any (one or more) of the criteria described above.

[0163] Accordingly, the invention provides any of the following, orcompositions (including pharmaceutical compositions) comprising any ofthe following: (a) antibody E3; (b) a fragment or a region of theantibody E3; (c) a light chain of the antibody E3 as shown in FIG. 1B;(c) a heavy chain of the antibody E3 as shown in FIG. 1A; (d) one ormore variable region(s) from a light chain and/or a heavy chain of theantibody E3; (e) one or more CDR(s) (one, two, three, four, five or sixCDRs) of antibody E3 shown in FIGS. 1A and 1B; (f) CDR H3 from the heavychain of antibody E3 shown in FIG. 1A; (g) CDR L3 from the light chainof antibody E3 shown in FIG. 1B; (h) three CDRs from the light chain ofantibody E3 shown in FIG. 1B; (i) three CDRs from the heavy chain ofantibody E3 shown in FIG. 1A; (j) three CDRs from the light chain andthree CDRs from the heavy chain, of antibody E3 shown in FIGS. 1A and1B; and (k) an antibody comprising any one of (b) through (j). As isevident from the description herein, specifically excluded from theinvention are polypeptide embodiments consisting of the identical aminoacid sequence to an amino acid sequence of mouse monoclonal antibody,911. The extended CDR sequences of Mab 911 are shown in FIGS. 1A and 1B,and in SEQ ID NOS:9-14.

[0164] The CDR portions of antibody E3 (including Chothia and KabatCDRs) are diagrammatically depicted in FIGS. 1A and 1B, and consist ofthe following amino acid sequences: (a) heavy chain CDR 1 (“CDR H1”)GFSLIGYDLN (SEQ ID NO:3); (b) heavy chain CDR 2 (“CDR H2”)IIWGDGTTDYNSAVKS (SEQ ID NO:4); (c) heavy chain CDR 3 (“CDR H3”)GGYWYATSYYFDY (SEQ ID NO:5); (d) light chain CDR 1 (“CDR LI”)RASQSISNNLN (SEQ ID NO:6); (e) light chain CDR 2 (“CDR L2”) YTSRFHS (SEQID NO:7); and (f) light chain CDR 3 (“CDR L3”) QQEHTLPYT (SEQ ID NO:8).Determination of CDR regions is well within the skill of the art. It isunderstood that in some embodiments, CDRs can be a combination of theKabat and Chothia CDR (also termed “combined CDRs” or “extended CDRs”).In some embodiments, the CDRs comprise the Kabat CDR. In otherembodiments, the CDRs are the Chothia CDR.

[0165] In some embodiments, the invention provides an antibody whichcomprises at least one CDR that is substantially homologous to at leastone CDR, at least two, at least three, at least four, at least 5 CDRs ofE3 (or, in some embodiments substantially homologous to all 6 CDRs ofE3, or derived from E3). Other embodiments include antibodies which haveat least two, three, four, five, or six CDR(s) that are substantiallyhomologous to at least two, three, four, five or six CDRs of E3 orderived from E3. It is understood that, for purposes of this invention,binding specificity and/or overall activity (which may be in terms oftreating and/or preventing pain or inhibiting NGF-dependent survival ofE13.5 mouse trigeminal neurons) is generally retained, although theextent of activity may vary compared to E3 (may be greater or lesser).

[0166] The invention also provides a polypeptide (which may or may notbe an antibody) which comprises an amino acid sequence of E3 (shown inFIGS. 1A and 1B) that has any of the following: at least 5 contiguousamino acids, at least 8 contiguous amino acids, at least about 10contiguous amino acids, at least about 15 contiguous amino acids, atleast about 20 contiguous amino acids, at least about 25 contiguousamino acids, at least about 30 contiguous amino acids of a sequence ofE3, wherein at least 3 of the amino acids are from a variable region ofE3, with the understanding that embodiments that consist of theidentical amino acid sequence to an amino acid sequence of mousemonoclonal antibody, 911, are specifically excluded. The extended CDRsequences of Mab 911 are shown in FIGS. 1A and 1B, and in SEQ IDNOS:9-14. In one embodiment, the variable region is from a light chainof E3. In another embodiment, the variable region is from a heavy chainof E3. In another embodiment, the 5 (or more) contiguous amino acids arefrom a complementarity determining region (CDR) of E3 shown in FIGS. 1Aand 1B.

[0167] In another embodiment, the invention provides a polypeptide whichcomprises an amino acid sequence of E3 that has any of the following: atleast 5 contiguous amino acids, at least 8 contiguous amino acids, atleast about 10 contiguous amino acids, at least about 15 contiguousamino acids, at least about 20 contiguous amino acids, at least about 25contiguous amino acids, at least about 30 contiguous amino acids of asequence of E3, wherein the E3 sequence comprises any one or more of:amino acid residue L29 of CDRH1, 150 of CDRH2, W101 of CDRH3, and/orA103 of CDRH3; and/or amino acid residue S28 of CDRL1, N32 of CDRL1, T51of CDRL2, 91E of CDRL3 and/or H92 of CDRL3, with the understanding thatembodiments that consist of the identical amino acid sequence to anamino acid sequence of mouse monoclonal antibody, 911, are specificallyexcluded.

[0168] As is evident, throughout this disclosure, a sequential aminoacid numbering scheme is used to refer to amino acid residues in thevariable regions (that is, the amino acid residues in each variableregion are numbered in sequence). As is well known in the art, the Kabatand/or Chothia numbering systems are useful when comparing twoantibodies or polypeptides, such as an E3 antibody and an E3 variant (orpolypeptide suspected of being an E3 variant). It is well understood inthe art how to convert sequential numbering to Chothia and/or Kabatnumbering, if desired, for example, for use in making comparisonsbetween E3 and another polypeptide. FIG. 23 depicts the E3 variableregions numbered using sequential, Chothia and Kabat numbering. Inaddition, to facilitate comparison, generally it is understood thatframework residues generally, but not always, have approximately thesame number of residues. However, the CDRs may vary in size (i.e., it ispossible to have insertions and/or deletions of one or more amino acidresidues). When comparing an E3 antibody and a candidate E3 variant (forexample, in the case of a CDR region from a candidate sequence which islonger in the sequence in antibody E3 to which is is aligned), one mayfollow the following steps (though other methods are known in the art).The candidate antibody sequence is aligned with E3 antibody heavy chainand light chain variable regions. Alignment may be done by hand, or bycomputer using commonly accepted computer programs. Alignment may befacilitated by using some amino acid residues which are common to mostFab sequences. For example, the light and heavy chains each typicallyhave two cysteines, which are often found at a conserved position. It isunderstood that the amino acid sequence of a candidate variant antibodymay be longer (i.e. have inserted amino acid residues) or shorter (havedeleted amino acid residues). Suffixes may be added to the residuenumber to indicate the insertion of additional residues, e.g., residue34 abc. For candidate sequences which, for example, align with a E3sequence for, e.g., residues 33 and 35, but have no residue between themto align with residue 35, the residue 35 is simply not assigned to aresidue. In another approach, it is generally well known that comparisonmay be made between structural equivalent (e.g., same position in theantigen-antibody complex) amino acids when comparing CDRs of differentlengths. For example, the Chothia numbering (Al-Lazikani et al, supra)generally (but not in all cases), places insertions and deletions at thestructurally corresct positions. Structural equivalence may also bededuced or demonstrated using X-ray crystallography or double mutantcycle analysis (see Pons et al. (1999) Prot. Sci. 8:958-968).

[0169] The binding affinity of an anti-NGF antibody to NGF (such ashNGF) can be about 0.10 to about 0.80 nM, about 0.15 to about 0.75 nMand about 0.18 to about 0.72 nM. In some embodiments, the bindingaffinity is about 2 pM, about 5 pM, about 10 pM, about 15 pM, about 20pM, about 40 pM, or greater than about 40 pM. In one embodiment, thebinding affinity is between about 2 pM and 22 pM. In other embodiments,the binding affinity is less than about 10 nM, about 5 nM, about 4 nnM,about 3.5 nM, about 3 nM, about 2.5 nM, about 2 nM, about 1.5 nM, about1 nM, about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500pM, about 400 pM, about 300 pM, about 200 pM, about 150 pM, about 100pM, about 90 pM, about 80 pM, about 70 pM, about 60 pM, about 50 pM,about 40 pM, about 30 pM, about 10 pM. In some embodiments, the bindingaffinity is about 10 nM. In other embodiments, the binding affinity isless than about 10 nM. In other embodiments, the binding affinity isabout 0.1 nM or about 0.07 nM. In other nM embodiments, the bindingaffinity is less than about 0.1 nM or less than about 0.07 nM. In otherembodiments, the binding affinity is any of about 10 nM, about 5 nM,about 4 nM, about 3.5 nM, about 3 nM, about 2.5 nM, about 2 nM, about1.5 nM, about 1 nM, about 900 pM, about 800 pM, bout 700 pM, about 600pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about 150pM, about 100 pM, about 90 pM, about 80 pM, about 70 pM, about 60 pM,about 50 pM, about 40 pM, about 30 pM, about 10 pM to any of about 2 pM,about 5 pM, about 10 pM, about 15 pM, about 20 pM, or about 40 pM. Insome embodiments, the binding affinity is any of about 10 nM, about 5nM, about 4 mm, about 3.5 nM, about 3 nM, about 2.5 nM, about 2 nM,about 1.5 nM, about 1 nM, about 900 pM, about 800 pM, bout 700 pM, about600 pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about150 pM, about 100 pM, about 90 pM, about 80 pM, about 70 pM, about 60pM, about 50 pM, about 40 pM, about 30 pM, about 10 pM. In still otherembodiments, the binding affinity is about 2 pM, about 5 pM, about 10pM, about 15 pM, about 20 pM, about 40 pM, or greater than about 40 pM.

[0170] The binding affinity of the antibody to NGF can be determinedusing methods well known in the art. One way of determining bindingaffinity of antibodies to NGF is by measuring affinity of monofunctionalFab fragments of the antibody, as described in the Examples. To obtainmonofunctional Fab fragments, an antibody (for example, IgG) can becleaved with papain or expressed recombinantly. The affinity of ananti-NGF Fab fragment of an antibody can be determined by surfaceplasmon resonance (BIAcore3000™ surface plasmon resonance (SPR) system,BIAcore, INC, Piscaway N.J.), as described in the Examples. Thisprotocol is suitable for use in determining binding affinity of anantibody to NGF of any species, including human NGF, NGF of anothervertebrate (in some embodiments, mammalian) (such as mouse NGF, rat NGF,primate NGF), as well as for use with other neurotrophins, such as therelated neurotrophins NT3, NT4/5, and/or BDNF.

[0171] In some embodiments, the antibodies or peptides of the inventionmay inhibit (reduce, and/or block) human NGF-dependent survival of mouseE13.5 trigeminal neurons with an IC50 (in the presence of about 15 pM ofNGF) of about any of 200 pM, 150 pM, 100 pM, 80 pM, 60 pM, 40 pM, 20 pM,10 pM, or less. In some embodiments, the antibodies or peptides of theinvention may inhibit (reduce, and/or block) human NGF-dependentsurvival of mouse E13.5 trigeminal neurons with an IC50 (in the presenceof about 1.5 pM of NGF) of about any of 50 pM, 40 pM, 30 pM, 10 pM, 20pM, 10 pM, 5 pM, 2 pM, 1 pM, or less. In some embodiments, theantibodies or peptides of the invention may inhibit (reduce, and/orblock) rat NGF-dependent survival of mouse E13.5 trigeminal neurons withan IC50 (in the presence of about 15 pM of NGF) of about any of 150 pM,125 pM, 100 pM, 80 pM, 60 pM, 40 pM, 30 pM, 20 pM, 10 pM, 5 pM, or less.In some embodiments, the antibodies or peptides of the invention mayinhibit (reduce, and/or block) rat NGF-dependent survival of mouse E13.5trigeminal neurons with an IC50 (in the presence of about 1.5 pM of NGF)of about any of 30 pM, 25 pM, 20 pM, 15 pM, 10 pM, 5 pM, 4 pM, 3 pM, 2pM, 1 pM, or less. Methods for measurement of the NGF-dependent survivalof mouse E13 trigeminal neurons are known in the art, and described,e.g., in Example 2.

[0172] The invention also provides methods of making any of theseantibodies or polypeptides. The antibodies of this invention can be madeby procedures known in the art, some of which are illustrated in theExamples. The polypeptides can be produced by proteolytic or otherdegradation of the antibodies, by recombinant methods (i.e., single orfusion polypeptides) as described above or by chemical synthesis.Polypeptides of the antibodies, especially shorter polypeptides up toabout 50 amino acids, are conveniently made by chemical synthesis.Methods of chemical synthesis are known in the art and are commerciallyavailable. For example, a E3 antibody could be produced by an automatedpolypeptide synthesizer employing the solid phase method. See also, U.S.Pat. Nos. 5,807,715; 4,816,567; and 6,331,415. Chimeric or hybridantibodies also may be prepared in vitro using known methods ofsynthetic protein chemistry, including those involving cross-linkingagents. For example, immunotoxins may be constructed using a disulfideexchange reaction or by forming a thioether bond. Examples of suitablereagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

[0173] In another alternative, the antibodies can be made recombinantlyusing procedures that are well known in the art. In one embodiment, apolynucleotide comprising a sequence encoding the variable and lightchain regions of antibody E3 (shown in FIGS. 1A and 1B) is cloned into avector for expression or propagation in a host cell (e.g., CHO cells).In another embodiment, the polynucleotide sequences shown in FIGS. 2 and3 are cloned into one or more vectors for expression or propagation. Thesequence encoding the antibody of interest may be maintained in a vectorin a host cell and the host cell can then be expanded and frozen forfuture use. Vectors (including expression vectors) and host cells arefurther described herein. Methods for expressing antibodiesrecombinantly in plants or milk have been disclosed. See, for example,Peeters et al. (2001) Vaccine 19:2756; Lonberg, N. and D. Huszar (1995)Int. Rev. Immunol 13:65; and Pollock et al. (1999) J Immunol Methods231:147. Methods for making derivatives of antibodies, e.g., humanized,single chain, etc. are known in the art.

[0174] The invention also encompasses single chain variable regionfragments (“scFv”) of antibodies of this invention, such as E3. Singlechain variable region fragments are made by linking light and/or heavychain variable regions by using a short linking peptide. Bird et al.(1988) Science 242:423-426. An example of a linking peptide is (GGGGS)₃(SEQ ID NO:15), which bridges approximately 3.5 nm between the carboxyterminus of one variable region and the amino terminus of the othervariable region. Linkers of other sequences have been designed and used(Bird et al. (1988)). Linkers can in turn be modified for additionalfunctions, such as attachment of drugs or attachment to solid supports.The single chain variants can be produced either recombinantly orsynthetically. For synthetic production of scFv, an automatedsynthesizer can be used. For recombinant production of scFv, a suitableplasmid containing polynucleotide that encodes the scFv can beintroduced into a suitable host cell, either eukaryotic, such as yeast,plant, insect or mammalian cells, or prokaryotic, such as E. coli.Polynucleotides encoding the scFv of interest can be made by routinemanipulations such as ligation of polynucleotides. The resultant scFvcan be isolated using standard protein purification techniques known inthe art.

[0175] Other forms of single chain antibodies, such as diabodies arealso encompassed. Diabodies are bivalent, bispecific antibodies in whichVH and VL domains are expressed on a single polypeptide chain, but usinga linker that is too short to allow for pairing between the two domainson the same chain, thereby forcing the domains to pair withcomplementary domains of another chain and creating two antigen bindingsites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123).

[0176] The antibody may be a bispecific antibody, a monoclonal antibodythat has binding specificities for at least two different antigens. Abisecific antibody can be prepared using the antibodies disclosedherein. Methods for making bispecific antibodies are known in the art(see, e.g., Suresh et al., 1986, Methods in Enzymology 121:210).Traditionally, the recombinant production of bispecific antibodies wasbased on the coexpression of two immunoglobulin heavy chain-light chainpairs, with the two heavy chains having different specificities(Millstein and Cuello, 1983, Nature 305, 537-539).

[0177] According to one approach to making bispecific antibodies,antibody variable domains with the desired binding specificities(antibody-antigen combining sites) are fused to immunoglobulin constantdomain sequences. The fusion preferably is with an immunoglobulin heavychain constant domain, comprising at least part of the hinge, CH2 andCH3 regions. It is preferred to have the first heavy chain constantregion (CH1), containing the site necessary for light chain binding,present in at least one of the fusions. DNAs encoding the immunoglobulinheavy chain fusions and, if desired, the immunoglobulin light chain, areinserted into separate expression vectors, and are cotransfected into asuitable host organism. This provides for great flexibility in adjustingthe mutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance.

[0178] In one approach, the bispecific antibodies are composed of ahybrid immunoglobulin heavy chain with a first binding specificity inone arm, and a hybrid immunoglobulin heavy chain-light chain pair(providing a second binding specificity) in the other arm. Thisasymmetric structure, with an immunoglobulin light chain in only onehalf of the bispecific molecule, facilitates the separation of thedesired bispecific compound from unwanted immunoglobulin chaincombinations. This approach is described in PCT Publication No. WO94/04690, published Mar. 3, 1994.

[0179] Heteroconjugate antibodies, comprising two covalently joinedantibodies, are also within the scope of the invention. Such antibodieshave been used to target immune system cells to unwanted cells (U.S.Pat. No. 4,676,980), and for treatment of HIV infection (PCT applicationpublication Nos. WO 91/00360 and WO 92/200373; EP 03089).Heteroconjugate antibodies may be made using any convenientcross-linking methods. Suitable cross-linking agents and techniques arewell known in the art, and are described in U.S. Pat. No. 4,676,980.

[0180] The antibody may be a humanized antibody, for example, as knownin the art, and as described herein.

[0181] Antibodies may be modified as described in PCT Publication No. WO99/58572, published Nov. 18, 1999. These antibodies comprise, inaddition to a binding domain directed at the target molecule, aneffector domain having an amino acid sequence substantially homologousto all or part of a constant domain of a human immunoglobulin heavychain. These antibodies are capable of binding the target moleculewithout triggering significant complement dependent lysis, orcell-mediated destruction of the target. Preferably, the effector domainis capable of specifically binding FcRn and/or FcγRIIb. These aretypically based on chimeric domains derived from two or more humanimmunoglobulin heavy chain CH2 domains. Antibodies modified in thismanner are preferred for use in chronic antibody therapy, to avoidinflammatory and other adverse reactions to conventional antibodytherapy.

[0182] The invention encompasses modifications to antibody E3, includingfunctionally equivalent antibodies which do not significantly affecttheir properties and variants which have enhanced or decreased activity.Modification of polypeptides is routine practice in the art and isfurther exemplified in the Examples. Examples of modified polypeptidesinclude polypeptides with substitutions (including conservativesubstitutions) of amino acid residues, one or more deletions oradditions of amino acids which do not significantly deleteriously changethe functional activity, or use of chemical analogs.

[0183] A polypeptide “variant,” as used herein, is a polypeptide thatdiffers from a native protein in one or more substitutions, deletions,additions and/or insertions, such that the immunoreactivity of thepolypeptide is not substantially diminished. In other words, the abilityof a variant to specifically bind antigen may be enhanced or unchanged,relative to the native protein, or may be diminished by less than 50%,and preferably less than 20%, relative to the native protein.Polypeptide variants preferably exhibit at least about 80%, morepreferably at least about 90% and most preferably at least about 95%identity (determined as described herein) to the identifiedpolypeptides.

[0184] Amino acid sequence variants of the antibodies may be prepared byintroducing appropriate nucleotide changes into the antibody DNA, or bypeptide synthesis. Such variants include, for example, deletions from,and/or insertions into and/or substitutions of, residues within theamino acid sequences of SEQ ID NO: 1 or 2 described herein. Anycombination of deletion, insertion, and substitution is made to arriveat the final construct, provided that the final construct possesses thedesired characteristics. The amino acid changes also may alterpost-translational processes of the antibody, such as changing thenumber or position of glycosylation sites.

[0185] A useful method for identification of certain residues or regionsof the antibody that are preferred locations for mutagenesis ormodification is called “alanine scanning mutagenesis,” and is describedby Cunningham and Wells, 1989, Science, 244:1081-1085. A residue orgroup of target residues is identified (e.g., charged residues such asarg, asp, his, lys, and glu) and replaced by a neutral or negativelycharged amino acid (most preferably alanine or polyalanine) to affectthe interaction of the amino acids with antigen. Those amino acidlocations demonstrating functional sensitivity to the substitutions thenare refined by introducing further or other variants at, or for, thesites of substitution. Thus, while the site for introducing an aminoacid sequence variation is predetermined, the nature of the mutation perse need not be predetermined. For example, to analyze the performance ofa mutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressed antibodyvariants are screened for the desired activity. Library scanningmutagenesis, as described herein, may also be used to identify locationsin an antibody that are suitable for mutagenesis or modification.

[0186] Amino acid sequence insertions include amino- and/orcarboxyl-terminal fusions ranging in length from one residue topolypeptides containing a hundred or more residues, as well asintrasequence insertions of single or multiple amino acid residues.Examples of terminal insertions include an antibody with an N-terminalmethionyl residue or the antibody fused to an epitope tag. Otherinsertional variants of the antibody molecule include the fusion to theN- or C-terminus of the antibody of an enzyme or a polypeptide whichincreases the serum half-life of the antibody.

[0187] Substitution variants have at least one amino acid residue in theantibody molecule removed and a different residue inserted in its place.The sites of greatest interest for substitutional mutagenesis includethe hypervariable regions, but FR alterations are also contemplated.Conservative substitutions are shown in Table 1 under the heading of“conservative substitutions”. If such substitutions result in a changein biological activity, then more substantial changes, denominated“exemplary substitutions” in Table 1, or as further described below inreference to amino acid classes, may be introduced and the productsscreened. TABLE 1 Amino Acid Substitutions Original ConservativeExemplary Residue Substitutions Substitutions Ala (A) Val Val; Leu; IleArg (R) Lys Lys; Gln; Asn Asn (N) Gln Gln; His; Asp, Lys; Arg Asp (D)Glu Glu; Asn Cys (C) Ser Ser; Ala Gln (Q) Asn Asn; Glu Glu (E) Asp Asp;Gln Gly (G) Ala Ala His (H) Arg Asn; Gln; Lys; Arg Ile (I) Leu Leu; Val;Met; Ala; Phe; Norleucine Leu (L) Ile Norleucine; Ile; Val; Met; Ala;Phe Lys (K) Arg Arg; Gln; Asn Met (M) Leu Leu; Phe; Ile Phe (F) Tyr Leu;Val; Ile; Ala; Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp(W) Tyr Tyr; Phe Tyr (Y) Phe Trp; Phe; Thr; Ser Val (V) Leu Ile; Leu;Met; Phe; Ala; Norleucine

[0188] Substantial modifications in the biological properties of theantibody are accomplished by selecting substitutions that differsignificantly in their effect on maintaining (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties:

[0189] (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

[0190] (2) Neutral hydrophilic: Cys, Ser, Thr;

[0191] (3) Acidic: Asp, Glu;

[0192] (4) Basic: Asn, Gln, His, Lys, Arg;

[0193] (5) Residues that influence chain orientation: Gly, Pro; and

[0194] (6) Aromatic: Trp, Tyr, Phe.

[0195] Non-conservative substitutions are made by exchanging a member ofone of these classes for another class.

[0196] Any cysteine residue not involved in maintaining the properconformation of the antibody also may be substituted, generally withserine, to improve the oxidative stability of the molecule and preventaberrant cross-linking. Conversely, cysteine bond(s) may be added to theantibody to improve its stability, particularly where the antibody is anantibody fragment such as an Fv fragment.

[0197] Amino acid modifications can range from changing or modifying oneor more amino acids to complete redesign of a region, such as thevariable region. Changes in the variable region can alter bindingaffinity and/or specificity. In some embodiment, no more than one tofive conservative amino acid substitutions are made within a CDR domain.In other embodiments, no more than one to three conservative amino acidsubstitutions are made within a CDR3 domain. In still other embodiments,the CDR domain is CDRH3 and/or CDR L3.

[0198] Modifications also include glycosylated and nonglycosylatedpolypeptides, as well as polypeptides with other post-translationalmodifications, such as, for example, glycosylation with differentsugars, acetylation, and phosphorylation. Antibodies are glycosylated atconserved positions in their constant regions (Jefferis and Lund, 1997,Chem. Immunol. 65:111-128; Wright and Morrison, 1997, TibTECH 15:26-32).The oligosaccharide side chains of the immunoglobulins affect theprotein's function (Boyd et al., 1996, Mol. Immunol. 32:1311-1318;Wittwe and Howard, 1990, Biochem. 29:4175-4180) and the intramolecularinteraction between portions of the glycoprotein, which can affect theconformation and presented three-dimensional surface of the glycoprotein(Hefferis and Lund, supra; Wyss and Wagner, 1996, Current Opin. Biotech.7:409-416). Oligosaccharides may also serve to target a givenglycoprotein to certain molecules based upon specific recognitionstructures. Glycosylation of antibodies has also been reported to affectantibody-dependent cellular cytotoxicity (ADCC). In particular, CHOcells with tetracycline-regulated expression ofβ(1,4)-N-acetylglucosaminyltransferase III (GnTIII), aglycosyltransferase catalyzing formation of bisecting GlcNAc, wasreported to have improved ADCC activity (Umana et al., 1999, MatureBiotech. 17:176-180).

[0199] Glycosylation of antibodies is typically either N-linked orO-linked. N-linked refers to the attachment of the carbohydrate moietyto the side chain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

[0200] Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

[0201] The glycosylation pattern of antibodies may also be alteredwithout altering the underlying nucleotide sequence. Glycosylationlargely depends on the host cell used to express the antibody. Since thecell type used for expression of recombinant glycoproteins, e.g.antibodies, as potential therapeutics is rarely the native cell,variations in the glycosylation pattern of the antibodies can beexpected (see, e.g. Hse et al., 1997, J. Biol. Chem. 272:9062-9070).

[0202] In addition to the choice of host cells, factors that affectglycosylation during recombinant production of antibodies include growthmode, media formulation, culture density, oxygenation, pH, purificationschemes and the like. Various methods have been proposed to alter theglycosylation pattern achieved in a particular host organism includingintroducing or overexpressing certain enzymes involved inoligosaccharide production (U.S. Pat. Nos. 5,047,335; 5,510,261 and5,278,299). Glycosylation, or certain types of glycosylation, can beenzymatically removed from the glycoprotein, for example usingendoglycosidase H (Endo H). In addition, the recombinant host cell canbe genetically engineered to be defective in processing certain types ofpolysaccharides. These and similar techniques are well known in the art.

[0203] Other methods of modification include using coupling techniquesknown in the art, including, but not limited to, enzymatic means,oxidative substitution and chelation. Modifications can be used, forexample, for attachment of labels for immunoassay. Modified E3polypeptides are made using established procedures in the art and can bescreened using standard assays known in the art, some of which aredescribed below and in the Examples.

[0204] Other antibody modifications include antibodies that have beenmodified as described in PCT Publication No. WO 99/58572, published Nov.18, 1999. These antibodies comprise, in addition to a binding domaindirected at the target molecule, an effector domain having an amino acidsequence substantially homologous to all or part of a constant domain ofa human immunoglobulin heavy chain. These antibodies are capable ofbinding the target molecule without triggering significant complementdependent lysis, or cell-mediated destruction of the target. In someembodiments, the effector domain is capable of specifically binding FcRnand/or FcγRIIb. These are typically based on chimeric domains derivedfrom two or more human immunoglobulin heavy chain CH2 domains.Antibodies modified in this manner are particularly suitable for use inchronic antibody therapy, to avoid inflammatory and other adversereactions to conventional antibody therapy.

[0205] The invention also encompasses fusion proteins comprising one ormore fragments or regions from the antibodies (such as E3) orpolypeptides of this invention. In one embodiment, a fusion polypeptideis provided that comprises at least 10 contiguous amino acids of thevariable light chain region shown in FIG. 1B and/or at least 10 aminoacids of the variable heavy chain region shown in FIG. 1A. In anotherembodiment, the fusion polypeptide comprises a light chain variableregion and/or a heavy chain variable region of E3, as shown in FIGS. 1Aand 1B. In another embodiment, the fusion polypeptide comprises one ormore CDR(s) of E3. In still other embodiments, the fusion polypeptidecomprises CDR H3 and/or CDR L3 of antibody E3. In another embodiment,the fusion polypeptide comprises any one or more of: amino acid residueL29 of CDRH1, 150 of CDRH2, W101 of CDRH3, and/or A103 of CDRH3; and/oramino acid residue S28 of CDRL1, N32 of CDRL1, T51 of CDRL2, 91E ofCDRL3 and/or H92 of CDRL3. For purposes of this invention, a E3 fusionprotein contains one or more E3 antibodies and another amino acidsequence to which it is not attached in the native molecule, forexample, a heterologous sequence or a homologous sequence from anotherregion. Exemplary heterologous sequences include, but are not limited toa “tag” such as a FLAG tag or a 6His tag. Tags are well known in theart.

[0206] A E3 fusion polypeptide can be created by methods known in theart, for example, synthetically or recombinantly. Typically, the E3fusion proteins of this invention are made by preparing an expressing apolynucleotide encoding them using recombinant methods described herein,although they may also be prepared by other means known in the art,including, for example, chemical synthesis.

[0207] This invention also provides compositions comprising E3antibodies or polypeptides conjugated (for example, linked) to an agentthat facilitate coupling to a solid support (such as biotin or avidin).For simplicity, reference will be made generally to E3 or antibodieswith the understanding that these methods apply to any of the NGFbinding embodiments described herein. Conjugation generally refers tolinking these components as described herein. The linking (which isgenerally fixing these components in proximate association at least foradministration) can be achieved in any number of ways. For example, adirect reaction between an agent and an antibody is possible when eachpossesses a substituent capable of reacting with the other. For example,a nucleophilic group, such as an amino or sulfhydryl group, on one maybe capable of reacting with a carbonyl-containing group, such as ananhydride or an acid halide, or with an alkyl group containing a goodleaving group (e.g., a halide) on the other.

[0208] An antibody or polypeptide of this invention may be linked to alabeling agent (alternatively termed “label”) such as a fluorescentmolecule, a radioactive molecule or any others labels known in the art.Labels are known in the art which generally provide (either directly orindirectly) a signal. Accordingly, the invention includes labeledantibodies and polypeptides.

[0209] The ability of the antibodies and polypeptides of this invention,such as binding NGF; reducing or inhibiting a NGF biological activity;reducing and/or blocking NGF-induced survival of E13.5 mouse trigeminalneurons, may be tested using methods known in the art, some of which aredescribed in the Examples.

[0210] The invention also provides compositions (includingpharmaceutical compositions) and kits comprising antibody E3, and, asthis disclosure makes clear, any or all of the antibodies and/orpolypeptides described herein.

[0211] Polynucleotides, Vectors and Host Cells

[0212] The invention also provides isolated polynucleotides encoding theantibodies and polypeptides of the invention (including an antibodycomprising the polypeptide sequences of the light chain and heavy chainvariable regions shown in FIGS. 1A and 1B), and vectors and host cellscomprising the polynucleotide.

[0213] Accordingly, the invention provides polynucleotides (orcompositions, including pharmaceutical compositions), comprisingpolynucleotides encoding any of the following: (a) antibody E3; (b) afragment or a region of the antibody E3; (c) a light chain of theantibody E3 as shown in FIG. 1B; (d) a heavy chain of the antibody E3 asshown in FIG. 1A; (e) one or more variable region(s) from a light chainand/or a heavy chain of the antibody E3; (f) one or more CDR(s) (one,two, three, four, five or six CDRs) of antibody E3 shown in FIGS. 1A and11B; (g) CDR H3 from the heavy chain of antibody E3 shown in FIG. 1A;(h) CDR L3 from the light chain of antibody E3 shown in FIG. 1B; (i)three CDRs from the light chain of antibody E3 shown in FIG. 1B; (j)three CDRs from the heavy chain of antibody E3 shown in FIG. 1A; (k)three CDRs from the light chain and three CDRs from the heavy chain, ofantibody E3 shown in FIGS. 1A and 1B; or (1) an antibody comprising anyof (b) to (k). In some embodiments, the polynucleotide comprises eitheror both of the polynucleotide(s) shown in FIGS. 2 and 3.

[0214] In another aspect, the invention is an isolated polynucleotidethat encodes for an E3 light chain with a deposit number of ATCC No.PTA-4893 or ATCC No. PTA-4894. In another aspect, the invention is anisolated polynucleotide that encodes for an E3 heavy chain with adeposit number of ATCC No. PTA-4895. In yet another aspect, theinvention is an isolated polynucleotide comprising (a) a variable regionencoded in the polynucleotide with a deposit number of ATCC No. PTA-4894and (b) a variable region encoded in the polynucleotide with a depositnumber of ATCC No. PTA-4895. In another aspect, the invention is anisolated polynucleotide comprising (a) one or more CDR encoded in thepolynucleotide with a deposit number of ATCC No. PTA-4894; and/or (b)one or more CDR encoded in the polynucleotide with a deposit number ofATCC No. PTA-4895.

[0215] In another aspect, the invention provides polynucleotidesencoding any of the antibodies (including antibody fragments) andpolypeptides described herein. Polynucleotides can be made by proceduresknown in the art

[0216] In another aspect, the invention provides compositions (such as apharmaceutical compositions) comprising any of the polynucleotides ofthe invention. In some embodiments, the composition comprises anexpression vector comprising a polynucleotide encoding the E3 antibodyas described herein. In other embodiment, the composition comprises anexpression vector comprising a polynucleotide encoding any of theantibodies or polypeptides described herein. In still other embodiments,the composition comprises either or both of the polynucleotides shown inFIGS. 2 and 3. Expression vectors, and administration of polynucleotidecompositions are further described herein.

[0217] In another aspect, the invention provides a method of making anyof the polynucleotides described herein.

[0218] Polynucleotides complementary to any such sequences are alsoencompassed by the present invention. Polynucleotides may besingle-stranded (coding or antisense) or double-stranded, and may be DNA(genomic, cDNA or synthetic) or RNA molecules. RNA molecules includeHnRNA molecules, which contain introns and correspond to a DNA moleculein a one-to-one manner, and mRNA molecules, which do not containintrons. Additional coding or non-coding sequences may, but need not, bepresent within a polynucleotide of the present invention, and apolynucleotide may, but need not, be linked to other molecules and/orsupport materials.

[0219] Polynucleotides may comprise a native sequence (i.e., anendogenous sequence that encodes an antibody or a portion thereof) ormay comprise a variant of such a sequence. Polynucleotide variantscontain one or more substitutions, additions, deletions and/orinsertions such that the immunoreactivity of the encoded polypeptide isnot diminished, relative to a native immunoreactive molecule. The effecton the immunoreactivity of the encoded polypeptide may generally beassessed as described herein. Variants preferably exhibit at least about70% identity, more preferably at least about 80% identity and mostpreferably at least about 90% identity to a polynucleotide sequence thatencodes a native antibody or a portion thereof.

[0220] Two polynucleotide or polypeptide sequences are said to be“identical” if the sequence of nucleotides or amino acids in the twosequences is the same when aligned for maximum correspondence asdescribed below. Comparisons between two sequences are typicallyperformed by comparing the sequences over a comparison window toidentify and compare local regions of sequence similarity. A “comparisonwindow” as used herein, refers to a segment of at least about 20contiguous positions, usually 30 to about 75, 40 to about 50, in which asequence may be compared to a reference sequence of the same number ofcontiguous positions after the two sequences are optimally aligned.

[0221] Optimal alignment of sequences for comparison may be conductedusing the Megalign program in the Lasergene suite of bioinformaticssoftware (DNASTAR, Inc., Madison, Wis.), using default parameters. Thisprogram embodies several alignment schemes described in the followingreferences: Dayhoff, M. O. (1978) A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ., 1990, Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M., 1989, CABIOS 5:151-153; Myers, E. W.and Muller W., 1988, CABIOS 4:11-17; Robinson, E. D., 1971, Comb. Theor.11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4:406-425; Sneath,P. H. A. and Sokal, R. R., 1973, Numerical Taxonomy the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J., 1983, Proc. Natl. Acad. Sci. USA80:726-730.

[0222] Preferably, the “percentage of sequence identity” is determinedby comparing two optimally aligned sequences over a window of comparisonof at least 20 positions, wherein the portion of the polynucleotide orpolypeptide sequence in the comparison window may comprise additions ordeletions (i.e. gaps) of 20 percent or less, usually 5 to 15 percent, or10 to 12 percent, as compared to the reference sequences (which does notcomprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid bases or amino acidresidue occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the reference sequence (i.e. the window size) andmultiplying the results by 100 to yield the percentage of sequenceidentity.

[0223] Variants may also, or alternatively, be substantially homologousto a native gene, or a portion or complement thereof. Suchpolynucleotide variants are capable of hybridizing under moderatelystringent conditions to a naturally occurring DNA sequence encoding anative antibody (or a complementary sequence).

[0224] Suitable “moderately stringent conditions” include prewashing ina solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50°C.-65° C., 5×SSC, overnight; followed by washing twice at 65° C. for 20minutes with each of 2×, 0.5× and 0.2×SSC containing 0.1% SDS.

[0225] As used herein, “highly stringent conditions” or “high stringencyconditions” are those that: (1) employ low ionic strength and hightemperature for washing, for example 0.015 M sodium chloride/0.0015 Msodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ duringhybridization a denaturing agent, such as formamide, for example, 50%(v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMsodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50%formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution,sonicated salmon sperm DNA (50 pg/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodiumcitrate) and 50% formamide at 55° C., followed by a high-stringency washconsisting of 0.1×SSC containing EDTA at 55° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

[0226] It will be appreciated by those of ordinary skill in the artthat, as a result of the degeneracy of the genetic code, there are manynucleotide sequences that encode a polypeptide as described herein. Someof these polynucleotides bear minimal homology to the nucleotidesequence of any native gene. Nonetheless, polynucleotides that vary dueto differences in codon usage are specifically contemplated by thepresent invention. Further, alleles of the genes comprising thepolynucleotide sequences provided herein are within the scope of thepresent invention. Alleles are endogenous genes that are altered as aresult of one or more mutations, such as deletions, additions and/orsubstitutions of nucleotides. The resulting mRNA and protein may, butneed not, have an altered structure or function. Alleles may beidentified using standard techniques (such as hybridization,amplification and/or database sequence comparison).

[0227] The polynucleotides of this invention can be obtained usingchemical synthesis, recombinant methods, or PCR. Methods of chemicalpolynucleotide synthesis are well known in the art and need not bedescribed in detail herein. One of skill in the art can use thesequences provided herein and a commercial DNA synthesizer to produce adesired DNA sequence.

[0228] For preparing polynucleotides using recombinant methods, apolynucleotide comprising a desired sequence can be inserted into asuitable vector, and the vector in turn can be introduced into asuitable host cell for replication and amplification, as furtherdiscussed herein. Polynucleotides may be inserted into host cells by anymeans known in the art. Cells are transformed by introducing anexogenous polynucleotide by direct uptake, endocytosis, transfection,F-mating or electroporation. Once introduced, the exogenouspolynucleotide can be maintained within the cell as a non-integratedvector (such as a plasmid) or integrated into the host cell genome. Thepolynucleotide so amplified can be isolated from the host cell bymethods well known within the art. See, e.g., Sambrook et al. (1989).

[0229] Alternatively, PCR allows reproduction of DNA sequences. PCRtechnology is well known in the art and is described in U.S. Pat. Nos.4,683,195, 4,800,159, 4,754,065 and 4,683,202, as well as PCR: ThePolymerase Chain Reaction, Mullis et al. eds., Birkauswer Press, Boston(1994).

[0230] RNA can be obtained by using the isolated DNA in an appropriatevector and inserting it into a suitable host cell. When the cellreplicates and the DNA is transcribed into RNA, the RNA can then beisolated using methods well known to those of skill in the art, as setforth in Sambrook et al., (1989), for example.

[0231] Suitable cloning vectors may be constructed according to standardtechniques, or may be selected from a large number of cloning vectorsavailable in the art. While the cloning vector selected may varyaccording to the host cell intended to be used, useful cloning vectorswill generally have the ability to self-replicate, may possess a singletarget for a particular restriction endonuclease, and/or may carry genesfor a marker that can be used in selecting clones containing the vector.Suitable examples include plasmids and bacterial viruses, e.g., pUC18,pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mpl8, mpl9,pBR322, pMB9, ColE1, pCR1, RP4, phage DNAs, and shuttle vectors such aspSA3 and pAT28. These and many other cloning vectors are available fromcommercial vendors such as BioRad, Strategene, and Invitrogen.

[0232] Expression vectors generally are replicable polynucleotideconstructs that contain a polynucleotide according to the invention. Itis implied that an expression vector must be replicable in the hostcells either as episomes or as an integral part of the chromosomal DNA.Suitable expression vectors include but are not limited to plasmids,viral vectors, including adenoviruses, adeno-associated viruses,retroviruses, cosmids, and expression vector(s) disclosed in PCTPublication No. WO 87/04462. Vector components may generally include,but are not limited to, one or more of the following: a signal sequence;an origin of replication; one or more marker genes; suitabletranscriptional controlling elements (such as promoters, enhancers andterminator). For expression (i.e., translation), one or moretranslational controlling elements are also usually required, such asribosome binding sites, translation initiation sites, and stop codons.

[0233] The vectors containing the polynucleotides of interest can beintroduced into the host cell by any of a number of appropriate means,including electroporation, transfection employing calcium chloride,rubidium chloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; and infection (e.g., where thevector is an infectious agent such as vaccinia virus). The choice ofintroducing vectors or polynucleotides will often depend on features ofthe host cell.

[0234] The invention also provides host cells comprising any of thepolynucleotides described herein. Any host cells capable ofover-expressing heterologous DNAs can be used for the purpose ofisolating the genes encoding the antibody, polypeptide or protein ofinterest. Non-limiting examples of mammalian host cells include but notlimited to COS, HeLa, and CHO cells. See also PCT Publication No. WO87/04462. Suitable non-mammalian host cells include prokaryotes (such asE. coli or B. subtillis) and yeast (such as S. cerevisae, S. pombe; orK. lactis). Preferably, the host cells express the cDNAs at a level ofabout 5 fold higher, more preferably 10 fold higher, even morepreferably 20 fold higher than that of the corresponding endogenousantibody or protein of interest, if present, in the host cells.Screening the host cells for a specific binding to NGF is effected by animmunoassay or FACS. A cell overexpressing the antibody or protein ofinterest can be identified.

[0235] Methods Using E3 and E3 Derived Antibodies

[0236] Antibody E3 which binds NGF may be used to identify or detect thepresence or absence of NGF. For simplicity, reference will be madegenerally to E3 or antibodies with the understanding that these methodsapply to any of the NGF binding embodiments (such as polypeptides)described herein. Detection generally involves contacting a biologicalsample with an antibody described herein that binds to NGF and theformation of a complex between NGF and an antibody (e.g., E3) whichbinds specifically to NGF. The formation of such a complex can be invitro or in vivo. The term “detection” as used herein includesqualitative and/or quantitative detection (measuring levels) with orwithout reference to a control.

[0237] Any of a variety of known methods can be used for detection,including, but not limited to, immunoassay, using antibody that bindsthe polypeptide, e.g. by enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA) and the like; and functional assay for theencoded polypeptide, e.g. binding activity or enzymatic assay. In someembodiments, the antibody is detectably labeled.

[0238] Diagnostic Uses of the E3 and Derivatives

[0239] Antibodies and polypeptides of the invention can be used in thedetection, diagnosis and monitoring of a disease, condition, or disorderassociated with altered or aberrant NGF expression (in some embodiments,increased or decreased NGF expression (relative to a normal sample),and/or inappropriate expression, such as presence of expression intissue(s) and/or cell(s) that normally lack NGF expression, or absenceof NGF expression in tissue(s) or cell(s) that normally possess NGFexpression). The antibodies and polypeptides of the invention arefurther useful for detection of NGF expression, for example, in adisease associated with altered or aberrant sensitivity orresponsiveness to NGF. In some embodiments, NGF expression is detectedin a sample from an individual suspected of having a disease, disorderfeaturing or associated with an altered or aberrant sensitivity orresponsiveness to NGF expression (e.g., a cancer in which NGF promotesgrowth and/or metastasis).

[0240] Thus, in some embodiments, the invention provides methodscomprising contacting a specimen (sample) of an individual suspected ofhaving altered or aberrant NGF expression with an antibody orpolypeptide of the invention and determining whether the level of NGFdiffers from that of a control or comparison specimen. In someembodiments, the individual has a cardiac arrhythmia, Alzheimer'sdisease, and/or autonomic dysfunction.

[0241] In other embodiments, the invention provides methods comprisescontacting a specimen (sample) of an individual and determining level ofNGF expression. In some embodiments, the individual is suspected ofhaving a disease, disorder featuring or associated with an altered oraberrant sensitivity or responsiveness to NGF expression. In someembodiments, the individual has small cell lung cancer, breast cancer,pancreatic cancer, prostate cancer, ovarian carcinoma, hepatocellularcarcinoma, or melanoma.

[0242] For diagnostic applications, the antibody typically will belabeled with a detectable moiety including but not limited toradioisotopes, fluorescent labels, and various enzyme-substrate labels.Methods of conjugating labels to an antibody are known in the art. Inother embodiment of the invention, antibodies of the invention need notbe labeled, and the presence thereof can be detected using a labeledantibody which binds to the antibodies of the invention.

[0243] The antibodies of the present invention may be employed in anyknown assay method, such competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies: A Manual of Techniques, pp.147-158 (CRC Press, Inc. 1987).

[0244] The antibodies may also be used for in vivo diagnostic assays,such as in vivo imaging. Generally, the antibody is labeled with aradionuclide (such as ¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ¹²⁵I, or ³H) so that thecells or tissue of interest can be localized using immunoscintiography.

[0245] The antibody may also be used as staining reagent in pathology,following techniques well known in the art.

[0246] Methods of Using E3 and Derivatives for Therapeutic Purposes

[0247] Antibody E3 is useful for reducing and/or blocking the biologicalactivity of NGF. This antagonistic activity is believed to be useful inthe treatment of pathological conditions associated with endogenous NGFproduction, such as pain. Generally, in these embodiments an effectiveamount is administered to an individual. Accordingly, in one aspect, theinvention provides a method of antagonizing human NGF biologicalactivity using any of the polypeptides (including antibodies such asantibody E3) disclosed herein. In one embodiment, the method comprisescontacting human nerve growth factor with any of the polypeptides(including antibody E3) described herein, whereby human nerve growthfactor activity is antagonized, reduced, blocked, or suppressed. In yetanother embodiment, an individual with pain (such as post-surgical pain,or rheumatoid arthritis pain) is given treatment with E3.

[0248] For simplicity, reference will be made generally to E3 orantibody with the understanding that these methods apply to any of theE3 variant antibodies and polypeptides described herein.

[0249] Various formulations of E3 or fragments of E3 (e.g., Fab, Fab′,F(ab′)₂, Fv, Fc, etc.), such as single chain (ScFv), mutants thereof,fusion proteins comprising an antibody portion, and any other modifiedconfiguration of E3 that comprises an antigen NGF recognition site ofthe required specificity, may be used for administration. In someembodiments, E3 antibodies or various formulations of E3 thereof may beadministered neat. In other embodiments, E3 or various formulations ofE3 (including any composition embodiment described herein) thereof and apharmaceutically acceptable excipient are administered, and may be invarious formulations. Pharmaceutically acceptable excipients are knownin the art, and are relatively inert substances that facilitateadministration of a pharmacologically effective substance. For example,an excipient can give form or consistency, or act as a diluent. Suitableexcipients include but are not limited to stabilizing agents, wettingand emulsifying agents, salts for varying osmolarity, encapsulatingagents, buffers, and skin penetration enhancers. Excipients as well asformulations for parenteral and nonparenteral drug delivery are setforth in Remington, The Science and Practice of Pharmacy 20th Ed. MackPublishing (2000).

[0250] In some embodiments, these agents are formulated foradministration by injection (e.g., intraperitoneally, intravenously,subcutaneously, intramuscularly, etc.), although other forms ofadministration (e.g., oral, mucosal, via inhalation, sublingually, etc)can be also used. Accordingly, E3 antibody and equivalents thereof arepreferably combined with pharmaceutically acceptable vehicles such assaline, Ringer's solution, dextrose solution, and the like. Theparticular dosage regimen, i.e., dose, timing and repetition, willdepend on the particular individual and that individual's medicalhistory. Generally, any of the following doses may be used: a dose of atleast about 50 mg/kg body weight; at least about 10 mg/kg body weight;at least about 3 mg/kg body weight; at least about 1 mg/kg body weight;at least about 750 μg/kg body weight; at least about 500 μg/kg bodyweight; at least about 250 μg/kg body weight; at least about 100 μg/kgbody weight; at least about 50 μg/kg body weight; at least about 10μg/kg body weight; at least about 1 μg/kg body weight, or less, isadministered. For repeated administrations over several days or longer,depending on the condition, the treatment is sustained until a desiredsuppression of disease symptoms occurs. An exemplary dosing regimencomprises administering an initial dose of about 2 mg/kg, followed by aweekly maintenance dose of about 1 mg/kg of the anti-NGF antibody, orfollowed by a maintenance dose of about 1 mg/kg every other week.However, other dosage regimens may be useful, depending on the patternof pharmacokinetic decay that the practitioner wishes to achieve.Empirical considerations, such as the half-life, generally willcontribute to determination of the dosage. The progress of this therapyis easily monitored by conventional techniques and assays.

[0251] In some individuals, more than one dose may be required.Frequency of administration may be determined and adjusted over thecourse of therapy. For example, frequency of administration may bedetermined or adjusted based on the type and severity of the pain to betreated, whether the agent is administered for preventive or therapeuticpurposes, previous therapy, the patient's clinical history and responseto the agent, and the discretion of the attending physician. Typicallythe clinician will administer an anti-NGF antagonist antibody (such asE3), until a dosage is reached that achieves the desired result. In somecases, sustained continuous release formulations of E3 antibodies may beappropriate. Various formulations and devices for achieving sustainedrelease are known in the art.

[0252] In one embodiment, dosages for E3 antibodies (or polypeptides)may be determined empirically in individuals who have been given one ormore administration(s). Individuals are given incremental dosages of E3.To assess efficacy of E3 or other equivalent antibody, markers of thedisease symptoms (such as pain) can be monitored.

[0253] Administration of an antibody (such as E3) or polypeptide inaccordance with the method in the present invention can be continuous orintermittent, depending, for example, upon the recipient's physiologicalcondition, whether the purpose of the administration is therapeutic orprophylactic, and other factors known to skilled practitioners. Theadministration of an antibody may be essentially continuous over apreselected period of time or may be in a series of spaced dose, e.g.,either before, during, or after developing pain, before, during, beforeand after, during and after, or before, during, and after developingpain. Administration can be before, during and/or after wound, incision,trauma, surgery, and any other event likely to give rise topost-surgical pain.

[0254] Other formulations include suitable delivery forms known in theart including, but not limited to, carriers such as liposomes. See, forexample, Mahato et al. (1997) Pharm. Res. 14:853-859. Liposomalpreparations include, but are not limited to, cytofectins, multilamellarvesicles and unilamellar vesicles.

[0255] In some embodiments, more than one antibody or polypeptide may bepresent. The antibodies can be monoclonal or polyclonal. Suchcompositions may contain at least one, at least two, at least three, atleast four, at least five different antibodies. A mixture of antibodies,as they are often denoted in the art, may be particularly useful intreating a broader range of population of individuals.

[0256] A polynucleotide encoding any of the antibodies or polypeptidesof the invention (such as antibody E3) may also be used for delivery andexpression of any of the antibodies or polypeptides of the invention(such as antibody E3) in a desired cell. It is apparent that anexpression vector can be used to direct expression of an E3 antibody orpolypeptide. The expression vector can be administered by any meansknown in the art, such as intraperitoneally, intravenously,intramuscularly, subcutaneously, intrathecally, intraventricularly,orally, enterally, parenterally, intranasally, dermally, sublingually,or by inhalation. For example, administration of expression vectorsincludes local or systemic administration, including injection, oraladministration, particle gun or catheterized administration, and topicaladministration. One skilled in the art is familiar with administrationof expression vectors to obtain expression of an exogenous protein invivo. See, e.g., U.S. Pat. Nos. 6,436,908; 6,413,942; and 6,376,471.

[0257] Targeted delivery of therapeutic compositions comprising apolynucleotide encoding any of the antibodies or polypeptides of theinvention (such as antibody E3) can also be used. Receptor-mediated DNAdelivery techniques are described in, for example, Findeis et al.,Trends Biotechnol. (1993) 11:202; Chiou et al., Gene Therapeutics:Methods And Applications Of Direct Gene Transfer (J. A. Wolff, ed.)(1994); Wu et al., J. Biol. Chem. (1988) 263:621; Wu et al., J. Biol.Chem. (1994) 269:542; Zenke et al., Proc. Natl. Acad. Sci. (USA) (1990)87:3655; Wu et al., J. Biol. Chem. (1991) 266:338. Therapeuticcompositions containing a polynucleotide are administered in a range ofabout 100 ng to about 200 mg of DNA for local administration in a genetherapy protocol. Concentration ranges of about 500 ng to about 50 mg,about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg toabout 100 μg of DNA can also be used during a gene therapy protocol. Thetherapeutic polynucleotides and polypeptides of the present inventioncan be delivered using gene delivery vehicles. The gene delivery vehiclecan be of viral or non-viral origin (see generally, Jolly, Cancer GeneTherapy (1994) 1:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly,Human Gene Therapy (1995) 1:185; and Kaplitt, Nature Genetics (1994)6:148). Expression of such coding sequences can be induced usingendogenous mammalian or heterologous promoters. Expression of the codingsequence can be either constitutive or regulated.

[0258] Viral-based vectors for delivery of a desired polynucleotide andexpression in a desired cell are well known in the art. Exemplaryviral-based vehicles include, but are not limited to, recombinantretroviruses (see, e.g., PCT Publication Nos. WO 90/07936; WO 94/03622;WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S.Pat. Nos. 5,219,740; 4,777,127; GB Patent No. 2,200,651; and EP PatentNo. 0 345 242), alphavirus-based vectors (e.g., Sindbis virus vectors,Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCCVR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCCVR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), and adeno-associatedvirus (AAV) vectors (see, e.g., PCT Publication Nos. WO 94/12649, WO93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655).Administration of DNA linked to killed adenovirus as described inCuriel, Hum. Gene Ther. (1992) 3:147 can also be employed.

[0259] Non-viral delivery vehicles and methods can also be employed,including, but not limited to, polycationic condensed DNA linked orunlinked to killed adenovirus alone (see, e.g., Curiel, Hum. Gene Ther.(1992) 3:147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989)264:16985); eukaryotic cell delivery vehicles cells (see, e.g., U.S.Pat. No. 5,814,482; PCT Publication Nos. WO 95/07994; WO 96/17072; WO95/30763; and WO 97/42338) and nucleic charge neutralization or fusionwith cell membranes. Naked DNA can also be employed. Exemplary naked DNAintroduction methods are described in PCT Publication No. WO 90/11092and U.S. Pat. No. 5,580,859. Liposomes that can act as gene deliveryvehicles are described in U.S. Pat. No. 5,422,120; PCT Publication Nos.WO 95/13796; WO 94/23697; WO 91/14445; and EP Patent NO. 0 524 968.Additional approaches are described in Philip, Mol. Cell Biol. (1994)14:2411 and in Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.

[0260] With respect to all methods described herein, reference toanti-NGF antagonist antibodies also include compositions comprising oneor more of these agents. These compositions may further comprisesuitable excipients, such as pharmaceutically acceptable excipientsincluding buffers, which are well known in the art. The presentinvention can be used alone or in combination with other conventionalmethods of treatment.

[0261] Methods of Using Anti-NGF Antagonist Antibody for Treating orPreventing Rheumatoid Arthritis Pain

[0262] In some aspects, the invention provides methods for treatingand/or preventing rheumatoid arthritis pain in individuals includingmammals, both human and non-human. Accordingly, in one aspect, theinvention provides methods of treating rheumatoid arthritis pain in anindividual comprising administering an effective amount of an anti-NGFantagonist antibody. Anti-NGF antagonist antibodies are known in the artand described herein.

[0263] In another aspect, the invention provides methods for reducingincidence of, ameliorating, suppressing, palliating, and/or delaying theonset, the development or the progression of rheumatoid arthritis painin an individual. Thus, in some embodiments, the anti-NGF antagonistantibody is administered prior to development of pain or a pain episodein an individual having rheumatoid arthritis.

[0264] In another aspect, the invention provides methods for treatinginflammatory cachexia (weight loss) associated with rheumatoid arthritisin an individual comprising administering an effective amount of ananti-NGF antagonist antibody (Roubenoff et al., Arthritis Rheum. 40(3):534-9 (1997); Roubenoff et al., J. Clin. Invest. 93(6):2379-86 (1994)).

[0265] Diagnosis or assessment of rheumatoid arthritis pain iswell-established in the art. Assessment may be performed based onmeasures known in the art, such as patient characterization of painusing various pain scales. See, e.g., Katz et al, Surg Clin North Am.(1999) 79 (2):231-52; Caraceni et al. J Pain Symptom Manage (2002)23(3):239-55. There are also commonly used scales to measure diseasestate such as the American College of Rheumatology (ACR) (Felson, etal., Arthritis and Rheumatism (1993) 36(6):729-740), the HealthAssessment Questionnaire (HAQ) (Fries, et al., (1982) J. Rheumatol. 9:789-793), the Paulus Scale (Paulus, et al., Arthritis and Rheumatism(1990) 33: 477-484), and the Arthritis Impact Measure Scale (AIMS)(Meenam, et al., Arthritis and Rheumatology (1982) 25: 1048-1053).Anti-NGF antagonist antibody may be administered to an individual viaany suitable route. Examples of different administration route aredescribed herein.

[0266] Pain relief may be characterized by time course of relief.Accordingly, in some embodiments, pain relief is observed within about24 hours after administration of anti-NGF antagonist antibody. In otherembodiments, pain relief is observed within about 36, 48, 60, 72 hoursor 4 days after administration of anti-NGF antagonist antibody. In stillother embodiments, pain relief is observed before observing anindication of improvement of the inflammatory condition associated withrheumatoid arthritis. In some embodiments, frequency and/or intensity ofpain is diminished, and/or quality of life of those suffering the thedisease is increased.

[0267] Making and using anti-NGF antibodies for these methods isdescribed in sections below (“Anti-NGF antagonist antibody”;“Identification of anti-NGF antagonist antibodies”; “Administration ofan anti-NGF antagonist antibody”).

[0268] Methods of Using Anti-NGF Antagonist Antibody For Treating orPreventing Osteoarthritis Pain

[0269] In some aspects, the invention provides methods for treatingand/or preventing osteoarthritis pain in individuals including mammals,both human and non-human. Accordingly, in one aspect, the inventionprovides methods of treating osteoarthritis pain in an individualcomprising administering an effective amount of an anti-NGF antagonistantibody. Anti-NGF antagonist antibodies are known in the art anddescribed herein.

[0270] In another aspect, the invention provides methods for reducingincidence of, ameliorating, suppressing, palliating, and/or delaying theonset, the development or the progression of osteoarthritis pain in anindividual. Thus, in some embodiments, the anti-NGF antagonist antibodyis administered prior to development of pain or a pain episode in anindividual having osteoarthritis.

[0271] Diagnosis or assessment of osteoarthritis pain iswell-established in the art. Assessment may be performed based onmeasures known in the art, such as patient characterization of painusing various pain scales. See, e.g., Katz et al, Surg Clin North Am.(1999) 79 (2):231-52; Caraceni et al. J Pain Symptom Manage (2002)23(3):239-55. For example, WOMAC Ambulation Pain Scale (including pain,stiffness, and physical function) and 100 mm Visual Analogue Scale (VAS)may be employed to assess pain and evaluate response to the treatment.

[0272] Anti-NGF antagonist antibody may be administered to an individualvia any suitable route. Examples of different administration route aredescribed herein.

[0273] Pain relief may be characterized by time course of relief.Accordingly, in some embodiments, pain relief is observed within about24 hours after administration of anti-NGF antagonist antibody. In otherembodiments, pain relief is observed within about 36, 48, 60, 72 hoursor 4 days after administration of anti-NGF antagonist antibody. In someembodiments, frequency and/or intensity of pain is diminished, and/orquality of life of those suffering the the disease is increased.

[0274] Making and using anti-NGF antibodies for these methods isdescribed in sections below (“Anti-NGF antagonist antibody”;“Identification of anti-NGF antagonist antibodies”; “Administration ofan anti-NGF antagonist antibody”).

[0275] Anti-NGF Antagonist Antibody

[0276] The methods of the invention (pertaining to rheumatoid arthritispain and osteoarthritis pain) use an anti-NGF antagonist antibody, whichrefers to any antibody molecule that blocks, suppresses or reduces(including significantly) NGF biological activity, including downstreampathways mediated by NGF signaling, such as receptor binding and/orelicitation of a cellular response to NGF.

[0277] An anti-NGF antagonist antibody should exhibit any one or more ofthe following characteristics: (a) bind to NGF and inhibit NGFbiological activity or downstream pathways mediated by NGF signalingfunction; (b) prevent, ameliorate, or treat any aspect of rheumatoidarthritis pain or osteoarthritis pain; (c) block or decrease NGFreceptor activation (including TrkA receptor dimerization and/orautophosphorylation); (d) increase clearance of NGF; (e) inhibit(reduce) NGF synthesis, production or release. Anti-NGF antagonistantibodies are known in the art, see, e.g., PCT Publication Nos. WO01/78698, WO 01/64247, U.S. Pat. Nos. 5,844,092, 5,877,016, and6,153,189; Hongo et al., Hybridoma, 19:215-227 (2000); Cell. Molec.Biol. 13:559-568 (1993); GenBank Accession Nos. U39608, U39609, L17078,or L17077.

[0278] For purposes of this invention, the antibody reacts with NGF in amanner that inhibits NGF and/or downstream pathways mediated by the NGFsignaling function. In some embodiments, the anti-NGF antagonistantibody recognizes human NGF. In yet other embodiments, the anti-NGFantagonist antibody specifically binds human NGF. In some embodiment,the anti-NGF antagonist antibody does not significantly bind to relatedneurotrophins, such as NT-3, NT4/5, and/or BDNF. In still otherembodiments, the anti-NGF antibody is capable of binding NGF andeffectively inhibiting the binding of NGF to its TrkA and/or p75receptor in vivo and/or effectively inhibiting NGF from activating itsTrkA and/or p75 receptor. In still other embodiment, the anti-NGFantagonist antibody is a monoclonal antibody. In still otherembodiments, the anti-NGF antibody is humanized (such as antibody E3described herein). In some embodiments, the anti-NGF antibody is human.In one embodiment, the antibody is a human antibody which recognizes oneor more epitopes on human NGF. In another embodiment, the antibody is amouse or rat antibody which recognizes one or more epitopes on humanNGF. In another embodiment, the antibody recognizes one or more epitopeson an NGF selected from the group consisting of: primate, canine,feline, equine, and bovine. In still further embodiments, the anti-NGFantagonist antibody binds essentially the same NGF epitope 6 as anantibody selected from any one or more of the following: MAb 911, MAb912 and MAb 938 (See Hongo, et al., Hybridoma 19:215-227 (2000)). Inother embodiments, the antibody binds the same epitope as Mab 911. Inanother embodiment, the antibody comprises a constant region that isimmunologically inert (e.g., does not trigger complement mediated lysisor antibody dependent cell mediated cytotoxicity (ADCC)). ADCC activitycan be assessed using methods disclosed in U.S. Pat. No. 5,500,362. Insome embodiments, the constant region is modified as described in Eur.J. Immunol. (1999) 29:2613-2624; PCT Application No. PCT/GB99/01441;and/or UK Patent Application No. 9809951.8.

[0279] In some embodiments, the anti-NGF antagonist antibody is ahumanized mouse anti-NGF monoclonal antibody termed antibody “E3”, anyof the E3 related antibodies described herein, or any fragments thereof,which are NGF antagonists.

[0280] The antibodies useful in the present invention can encompassmonoclonal antibodies, polyclonal antibodies, antibody fragments (e.g.,Fab, Fab′, F(ab′)₂, Fv, Fc, etc.), chimeric antibodies, bispecificantibodies, heteroconjugate antibodies, single chain (ScFv), mutantsthereof, fusion proteins comprising an antibody portion, humanizedantibodies, and any other modified configuration of the immunoglobulinmolecule that comprises an antigen recognition site of the requiredspecificity, including glycosylation variants of antibodies, amino acidsequence variants of antibodies, and covalently modified antibodies. Theantibodies may be murine, rat, human, or any other origin (includingchimeric or humanized antibodies).

[0281] The binding affinity of an anti-NGF antagonist antibody to NGF(such as hNGF) can be about 0.10 to about 0.80 nM, about 0.15 to about0.75 nM and about 0.18 to about 0.72 nM. In one embodiment, the bindingaffinity is between about 2 pM and 22 pM. In some embodiment, thebinding affinity is about 10 nM. In other embodiments, the bindingaffinity is less than about 10 nM. In other embodiments, the bindingaffinity is about 0.1 pM or about 0.07 nM. In other embodiments, thebinding affinity is less than about 0.1 nM or less than about 0.07 nM.In other embodiments, the binding affinity is any of about 100 pM, about50 nM, about 10 nM, about 1 nM, about 500 pM, about 100 pM, or about 50pM to any of about 2 pM, about 5 pM, about 10 pM, about 15 pM, about 20pM, or about 40 pM. In some embodiments, the binding affinity is any ofabout 100 nM, about 50 nM, about 10 nM, about 1 nM, about 500 pM, about100 pM, or about 50 pM, or less than about 50 pM. In some embodiments,the binding affinity is less than any of about 100 nM, about 50 nM,about 10 nM, about 1 nM, about 500 pM, about 100 pM, or about 50 pM. Instill other embodiments, the binding affinity is about 2 pM, about 5 pM,about 10 pM, about 15 pM, about 20 pM, about 40 pM, or greater thanabout 40 pM.

[0282] One way of determining binding affinity of antibodies to NGF isby measuring binding affinity of monofunctional Fab fragments of theantibody. To obtain monofunctional Fab fragments, an antibody (forexample, IgG) can be cleaved with papain or expressed recombinantly. Theaffinity of an anti-NGF Fab fragment of an antibody can be determined bysurface plasmon resonance (BIAcore3000™ surface plasmon resonance (SPR)system, BIAcore, INC, Piscaway N.J.). CM5 chips can be activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiinide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Human NGF (or any other NGF) can be diluted into 10 mM sodium acetate pH4.0 and injected over the activated chip at a concentration of 0.005mg/mL. Using variable flow time across the individual chip channels, tworanges of antigen density can be achieved: 100-200 response units (RU)for detailed kinetic studies and 500-600 RU for screening assays. Thechip can be blocked with ethanolamine. Regeneration studies have shownthat a mixture of Pierce elution buffer (Product No. 21004, PierceBiotechnology, Rockford Ill.) and 4 M NaCl (2:1) effectively removes thebound Fab while keeping the activity of hNGF on the chip for over 200injections. HBS-EP buffer (0.01M HEPES, pH 7.4, 0.15 NaCl, 3 mM EDTA,0.005% Surfactant P29) is used as running buffer for the BIAcore assays.Serial dilutions (0.1-10×estimated K_(D)) of purified Fab samples areinjected for 1 min at 100 μL/min and dissociation times of up to 2 h areallowed. The concentrations of the Fab proteins are determined by ELISAand/or SDS-PAGE electrophoresis using a Fab of known concentration (asdetermined by amino acid analysis) as a standard. Kinetic associationrates (k_(on)) and dissociation rates (k_(off)) are obtainedsimultaneously by fitting the data to a 1:1 Langmuir binding model(Karlsson, R. Roos, H. Fagerstam, L. Petersson, B. (1994). MethodsEnzymology 6. 99-110) using the BIAevaluation program. Equilibriumdissociation constant (K_(D)) values are calculated as k_(off)/k_(on).This protocol is suitable for use in determining binding affinity of anantibody to any NGF, including human NGF, NGF of another vertebrate (insome embodiments, mammalian) (such as mouse NGF, rat NGF, primate NGF),as well as for use with other neurotrophins, such as the relatedneurotrophins NT3, NT4/5, and/or BDNF. 1

[0283] In some embodiments, the antibody binds human NGF, and does notsignificantly bind an NGF from another vertebrate species (in someembodiment, mammalian). In some embodiments, the antibody binds humanNGF as well as one or more NGF from another vertebrate species (in someembodiments, mammalian). In still other embodiments, the antibody bindsNGF and does not significantly cross-react with other neurotrophins(such as the related neurotrophins, NT3, NT4/5, and/or BDNF). In someembodiments, the antibody binds NGF as well as at least one otherneurotrophin. In some embodiments, the antibody binds to a mammalianspecies of NGF, such as horse or dog, but does not significantly bind toNGF from anther mammalian species.

[0284] The epitope(s) can be continuous or discontinuous. In oneembodiment, the antibody binds essentially the same hNGF epitopes as anantibody selected from the group consisting of MAb 911, MAb 912, and MAb938 as described in Hongo et al., Hybridoma, 19:215-227 (2000). Inanother embodiment, the antibody binds essentially the same hNGF epitopeas MAb 911. In still another embodiment, the antibody binds essentiallythe same epitope as MAb 909. Hongo et al., supra. For example, theepitope may comprise one or more of: residues K32, K34 and E35 withinvariable region 1 (amino acids 23-35) of hNGF; residues F79 and T81within variable region 4 (amino acids 81-88) of hNGF; residues H84 andK88 within variable region 4; residue R103 between variable region 5(amino acids 94-98) of hNGF and the C-terminus (amino acids 111-118) ofhNGF; residue E11 within pre-variable region 1 (amino acids 10-23) ofhNGF; Y52 between variable region 2 (amino acids 40-49) of hNGF andvariable region 3 (amino acids 59-66) of hNGF; residues L112 and S113within the C-terminus of hNGF; residues R59 and R69 within variableregion 3 of hNGF; or residues V18, V20, and G23 within pre-variableregion 1 of hNGF. In addition, an epitope can comprise one or more ofthe variable region 1, variable region 3, variable region 4, variableregion 5, the N-terminus region, and/or the C-terminus of hNGF. In stillanother embodiment, the antibody significantly reduces the solventaccessibility of residue R103 of hNGF. It is understood that althoughthe epitopes described above relate to human NGF, one of ordinary skillcan align the structures of human NGF with the NGF of other species andidentify likely counterparts to these epitopes.

[0285] In one aspect, antibodies (e.g., human, humanized, mouse,chimeric) that can inhibit NGF may be made by using immunogens thatexpress full length or partial sequence of NGF. In another aspect, animmunogen comprising a cell that overexpresses NGF may be used. Anotherexample of an immunogen that can be used is NGF protein that containsfull-length NGF or a portion of the NGF protein.

[0286] The anti-NGF antagonist antibodies may be made by any methodknown in the art. The route and schedule of immunization of the hostanimal are generally in keeping with established and conventionaltechniques for antibody stimulation and production, as further describedherein. General techniques for production of human and mouse antibodiesare known in the art and are described herein.

[0287] It is contemplated that any mammalian subject including humans orantibody producing cells therefrom can be manipulated to serve as thebasis for production of mammalian, including human, hybridoma celllines. Typically, the host animal is inoculated intraperitoneally,intramuscularly, orally, subcutaneously, intraplantar, and/orintradermally with an amount of immunogen, including as describedherein.

[0288] Hybridomas can be prepared from the lymphocytes and immortalizedmyeloma cells using the general somatic cell hybridization technique ofKohler, B. and Milstein, C. (1975) Nature 256:495-497 or as modified byBuck, D. W., et al., In Vitro, 18:377-381 (1982). Available myelomalines, including but not limited to X63-Ag8.653 and those from the SalkInstitute, Cell Distribution Center, San Diego, Calif., USA, may be usedin the hybridization. Generally, the technique involves fusing myelomacells and lymphoid cells using a fusogen such as polyethylene glycol, orby electrical means well known to those skilled in the art. After thefusion, the cells are separated from the fusion medium and grown in aselective growth medium, such as hypoxanthine-aminopterin-thymidine(HAT) medium, to eliminate unhybridized parent cells. Any of the mediadescribed herein, supplemented with or without serum, can be used forculturing hybridomas that secrete monoclonal antibodies. As anotheralternative to the cell fusion technique, EBV immortalized B cells maybe used to produce the anti-NGF monoclonal antibodies of the subjectinvention. The hybridomas are expanded and subcloned, if desired, andsupernatants are assayed for anti-immunogen activity by conventionalimmunoassay procedures (e.g., radioimmunoassay, enzyme immunoassay, orfluorescence immunoassay).

[0289] Hybridomas that may be used as source of antibodies encompass allderivatives, progeny cells of the parent hybridomas that producemonoclonal antibodies specific for NGF, or a portion thereof.

[0290] Hybridomas that produce such antibodies may be grown in vitro orin vivo using known procedures. The monoclonal antibodies may beisolated from the culture media or body fluids, by conventionalimmunoglobulin purification procedures such as ammonium sulfateprecipitation, gel electrophoresis, dialysis, chromatography, andultrafiltration, if desired. Undesired activity if present, can beremoved, for example, by running the preparation over adsorbents made ofthe immunogen attached to a solid phase and eluting or releasing thedesired antibodies off the immunogen. Immunization of a host animal witha human NGF, or a fragment containing the target amino acid sequenceconjugated to a protein that is immunogenic in the species to beimmunized, e.g., keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, or soybean trypsin inhibitor using a bifunctional orderivatizing agent, for example maleimidobenzoyl sulfosuccinimide ester(conjugation through cysteine residues), N-hydroxysuccinimide (throughlysine residues), glytaradehyde, succinic anhydride, SOCl12, orR1N═C=NR, where R and R1 are different alkyl groups, can yield apopulation of antibodies (e.g., monoclonal antibodies).

[0291] If desired, the anti-NGF antagonist antibody (monoclonal orpolyclonal) of interest may be sequenced and the polynucleotide sequencemay then be cloned into a vector for expression or propagation. Thesequence encoding the antibody of interest may be maintained in vectorin a host cell and the host cell can then be expanded and frozen forfuture use. In an alternative, the polynucleotide sequence may be usedfor genetic manipulation to “humanize” the antibody or to improve theaffinity, or other characteristics of the antibody. For example, theconstant region may be engineered to more resemble human constantregions to avoid immune response if the antibody is used in clinicaltrials and treatments in humans. It may be desirable to geneticallymanipulate the antibody sequence to obtain greater affinity to NGF andgreater efficacy in inhibiting NGF. It will be apparent to one of skillin the art that one or more polynucleotide changes can be made to theanti-NGF antagonist antibody and still maintain its binding ability toNGF.

[0292] There are four general steps to humanize a monoclonal antibody.These are: (1) determining the nucleotide and predicted amino acidsequence of the starting antibody light and heavy variable domains (2)designing the humanized antibody, i.e., deciding which antibodyframework region to use during the humanizing process (3) the actualhumanizing methodologies/techniques and (4) the transfection andexpression of the humanized antibody. See, for example, U.S. Pat. Nos.4,816,567; 5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761;5,693,762; 5,585,089; and 6,180,370.

[0293] A number of “humanized” antibody molecules comprising anantigen-binding site derived from a non-human immunoglobulin have beendescribed, including chimeric antibodies having rodent or modifiedrodent V regions and their associated complementarity determiningregions (CDRs) fused to human constant domains. See, for example, Winteret al. Nature 349:293-299 (1991), Lobuglio et al. Proc. Nat. Acad. Sci.USA 86:4220-4224 (1989), Shaw et al. J. Immunol. 138:4534-4538 (1987),and Brown et al. Cancer Res. 47:3577-3583 (1987). Other referencesdescribe rodent CDRs grafted into a human supporting framework region(FR) prior to fusion with an appropriate human antibody constant domain.See, for example, Riechmann et al. Nature 332:323-327 (1988), Verhoeyenet al. Science 239:1534-1536 (1988), and Jones et al. Nature 321:522-525(1986). Another reference describes rodent CDRs supported byrecombinantly veneered rodent framework regions. See, for example,European Patent Publication No. 0519596. These “humanized” molecules aredesigned to minimize unwanted immunological response toward rodentanti-human antibody molecules which limits the duration andeffectiveness of therapeutic applications of those moieties in humanrecipients. For example, the antibody constant region can be engineeredsuch that it is immunologically inert (e.g., does not trigger complementlysis). See, e.g. PCT Publication No. PCT/GB99/01441; UK PatentApplication No. 9809951.8. Other methods of humanizing antibodies thatmay also be utilized are disclosed by Daugherty et al., Nucl. Acids Res.19:2471-2476 (1991) and in U.S. Pat. Nos. 6,180,377; 6,054,297;5,997,867; 5,866,692; 6,210,671; and 6,350,861; and in PCT PublicationNo. WO 01/27160.

[0294] In yet another alternative, fully human antibodies may beobtained by using commercially available mice that have been engineeredto express specific human immunoglobulin proteins. Transgenic animalsthat are designed to produce a more desirable (e.g., fully humanantibodies) or more robust immune response may also be used forgeneration of humanized or human antibodies. Examples of such technologyare Xenomouse™ from Abgenix, Inc. (Fremont, Calif.) and HuMAb-Mouse® andTC Mouse™ from Medarex, Inc. (Princeton, N.J.).

[0295] In an alternative, antibodies may be made recombinantly andexpressed using any method known in the art. In another alternative,antibodies may be made recombinantly by phage display technology. See,for example, U.S. Pat. Nos. 5,565,332; 5,580,717; 5,733,743; and6,265,150; and Winter et al., Annu. Rev. Immunol. 12:433-455 (1994).Alternatively, the phage display technology (McCafferty et al., Nature348:552-553 (1990)) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B cell. Phage display can be performed in avariety of formats; for review see, e.g., Johnson, Kevin S. andChiswell, David J., Current Opinion in Structural Biology 3:564-571(1993). Several sources of V-gene segments can be used for phagedisplay. Clackson et al., Nature 352:624-628 (1991) isolated a diversearray of anti-oxazolone antibodies from a small random combinatoriallibrary of V genes derived from the spleens of immunized mice. Arepertoire of V genes from unimmunized human donors can be constructedand antibodies to a diverse array of antigens (including self-antigens)can be isolated essentially following the techniques described by Market al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al., EMBO J.12:725-734 (1993). In a natural immune response, antibody genesaccumulate mutations at a high rate (somatic hypermutation). Some of thechanges introduced will confer higher affinity, and B cells displayinghigh-affinity surface immunoglobulin are preferentially replicated anddifferentiated during subsequent antigen challenge. This natural processcan be mimicked by employing the technique known as “chain shuffling.”Marks, et al., Bio/Technol. 10:779-783 (1992)). In this method, theaffinity of “primary” human antibodies obtained by phage display can beimproved by sequentially replacing the heavy and light chain V regiongenes with repertoires of naturally occurring variants (repertoires) ofV domain genes obtained from unimmunized donors. This technique allowsthe production of antibodies and antibody fragments with affinities inthe pM-nM range. A strategy for making very large phage antibodyrepertoires (also known as “the mother-of-all libraries”) has beendescribed by Waterhouse et al., Nucl. Acids Res. 21:2265-2266 (1993).Gene shuffling can also be used to derive human antibodies from rodentantibodies, where the human antibody has similar affinities andspecificities to the starting rodent antibody. According to this method,which is also referred to as “epitope imprinting”, the heavy or lightchain V domain gene of rodent antibodies obtained by phage displaytechnique is replaced with a repertoire of human V domain genes,creating rodent-human chimeras. Selection on antigen results inisolation of human variable regions capable of restoring a functionalantigen-binding site, i.e., the epitope governs (imprints) the choice ofpartner. When the process is repeated in order to replace the remainingrodent V domain, a human antibody is obtained (see PCT Publication No.WO 93/06213, published Apr. 1, 1993). Unlike traditional humanization ofrodent antibodies by CDR grafting, this technique provides completelyhuman antibodies, which have no framework or CDR residues of rodentorigin.

[0296] It is apparent that although the above discussion pertains tohumanized antibodies, the general principles discussed are applicable tocustomizing antibodies for use, for example, in dogs, cats, primate,equines and bovines. It is further apparent that one or more aspects ofhumanizing an antibody described herein may be combined, e.g., CDRgrafting, framework mutation and CDR mutation.

[0297] Antibodies may be made recombinantly by first isolating theantibodies and antibody producing cells from host animals, obtaining thegene sequence, and using the gene sequence to express the antibodyrecombinantly in host cells (e.g., CHO cells). Another method which maybe employed is to express the antibody sequence in plants (e.g.,tobacco) or transgenic milk. Methods for expressing antibodiesrecombinantly in plants or milk have been disclosed. See, for example,Peeters, et al. Vaccine 19:2756 (2001); Lonberg, N. and D. Huszar Int.Rev. Immunol 13:65 (1995); and Pollock, et al., J Immunol Methods231:147(1999). Methods for making derivatives of antibodies, e.g.,humanized, single chain, etc. are known in the art.

[0298] Immunoassays and flow cytometry sorting techniques such asfluorescence activated cell sorting (FACS) can also be employed toisolate antibodies that are specific for NGF.

[0299] The antibodies can be bound to many different carriers. Carrierscan be active and/or inert. Examples of well-known carriers includepolypropylene, polystyrene, polyethylene, dextran, nylon, amylases,glass, natural and modified celluloses, polyacrylamides, agaroses andmagnetite. The nature of the carrier can be either soluble or insolublefor purposes of the invention. Those skilled in the art will know ofother suitable carriers for binding antibodies, or will be able toascertain such, using routine experimentation. In some embodiments, thecarrier comprises a moiety that targets the myocardium.

[0300] DNA encoding the monoclonal antibodies is readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of the monoclonal antibodies). The hybridomacells serve as a preferred source of such DNA. Once isolated, the DNAmay be placed into expression vectors (such as expression vectorsdisclosed in PCT Publication No. WO 87/04462), which are thentransfected into host cells such as E. coli cells, simian COS cells,Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, to obtain the synthesis ofmonoclonal antibodies in the recombinant host cells. See, e.g., PCTPublication No. WO 87/04462. The DNA also may be modified, for example,by substituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences, Morrisonet al., Proc. Nat. Acad. Sci. 81:6851 (1984), or by covalently joiningto the immunoglobulin coding sequence all or part of the coding sequencefor a non-immunoglobulin polypeptide. In that manner, “chimeric” or“hybrid” antibodies are prepared that have the binding specificity of ananti-NGF monoclonal antibody herein.

[0301] Anti-NGF antagonist antibodies may be characterized using methodswell known in the art. For example, one method is to identify theepitope to which it binds, or “epitope mapping.” There are many methodsknown in the art for mapping and characterizing the location of epitopeson proteins, including solving the crystal structure of anantibody-antigen complex, competition assays, gene fragment expressionassays, and synthetic peptide-based assays, as described, for example,in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999. Inan additional example, epitope mapping can be used to determine thesequence to which an anti-NGF antagonist antibody binds. Epitope mappingis commercially available from various sources, for example, PepscanSystems (Edelhertweg 15, 8219 PH Lelystad, The Netherlands). The epitopecan be a linear epitope, i.e., contained in a single stretch of aminoacids, or a conformational epitope formed by a three-dimensionalinteraction of amino acids that may not necessarily be contained in asingle stretch. Peptides of varying lengths (e.g., at least 4-6 aminoacids long) can be isolated or synthesized (e.g., recombinantly) andused for binding assays with an anti-NGF antagonist antibody. In anotherexample, the epitope to which the anti-NGF antagonist antibody binds canbe determined in a systematic screening by using overlapping peptidesderived from the NGF sequence and determining binding by the anti-NGFantagonist antibody. According to the gene fragment expression assays,the open reading frame encoding NGF is fragmented either randomly or byspecific genetic constructions and the reactivity of the expressedfragments of NGF with the antibody to be tested is determined. The genefragments may, for example, be produced by PCR and then transcribed andtranslated into protein in vitro, in the presence of radioactive aminoacids. The binding of the antibody to the radioactively labeled NGFfragments is then determined by immunoprecipitation and gelelectrophoresis. Certain epitopes can also be identified by using largelibraries of random peptide sequences displayed on the surface of phageparticles (phage libraries). Alternatively, a defined library ofoverlapping peptide fragments can be tested for binding to the testantibody in simple binding assays. In an additional example, mutagenesisof an antigen binding domain, domain swapping experiments and alaninescanning mutagenesis can be performed to identify residues required,sufficient, and/or necessary for epitope binding. For example, domainswapping experiments can be performed using a mutant NGF in whichvarious fragments of the NGF polypeptide have been replaced (swapped)with sequences from a closely related, but antigenically distinctprotein (such as another member of the neurotrophin protein family). Byassessing binding of the antibody to the mutant NGF, the importance ofthe particular NGF fragment to antibody binding can be assessed.

[0302] Yet another method which can be used to characterize an anti-NGFantagonist antibody is to use competition assays with other antibodiesknown to bind to the same antigen, i.e., various fragments on NGF, todetermine if the anti-NGF antagonist antibody binds to the same epitopeas other antibodies. Competition assays are well known to those of skillin the art. Example of antibodies that can be used in the competitionassays for the present invention include MAb 911, 912, 938, as describedin Hongo, et al., Hybridoma 19:215-227 (2000).

[0303] An expression vector can be used to direct expression of ananti-NGF antagonist antibody. One skilled in the art is familiar withadministration of expression vectors to obtain expression of anexogenous protein in vivo. See, e.g., U.S. Pat. Nos. 6,436,908;6,413,942; and 6,376,471. Administration of expression vectors includeslocal or systemic administration, including injection, oraladministration, particle gun or catheterized administration, and topicaladministration. In another embodiment, the expression vector isadministered directly to the sympathetic trunk or ganglion, or into acoronary artery, atrium, ventricle, or pericardium.

[0304] Targeted delivery of therapeutic compositions containing anexpression vector, or subgenomic polynucleotides can also be used.Receptor-mediated DNA delivery techniques are described in, for example,Findeis et al., Trends Biotechnol. (1993) 11:202; Chiou et al., GeneTherapeutics: Methods And Applications Of Direct Gene Transfer (J. A.Wolff, ed.) (1994); Wu et al., J. Biol. Chem. (1988) 263:621; Wu et al.,J. Biol. Chem. (1994) 269:542; Zenke et al., Proc. Natl. Acad. Sci. USA(1990) 87:3655; Wu et al., J. Biol. Chem. (1991) 266:338. Therapeuticcompositions containing a polynucleotide are administered in a range ofabout 100 ng to about 200 mg of DNA for local administration in a genetherapy protocol. Concentration ranges of about 500 ng to about 50 mg,about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg toabout 100 μg of DNA can also be used during a gene therapy protocol. Thetherapeutic polynucleotides and polypeptides can be delivered using genedelivery vehicles. The gene delivery vehicle can be of viral ornon-viral origin (see generally, Jolly, Cancer Gene Therapy (1994) 1:51;Kimura, Human Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy(1995) 1:185; and Kaplitt, Nature Genetics (1994) 6:148). Expression ofsuch coding sequences can be induced using endogenous mammalian orheterologous promoters. Expression of the coding sequence can be eitherconstitutive or regulated.

[0305] Viral-based vectors for delivery of a desired polynucleotide andexpression in a desired cell are well known in the art. Exemplaryviral-based vehicles include, but are not limited to, recombinantretroviruses (see, e.g., PCT Publication Nos. WO 90/07936; WO 94/03622;WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S.Pat. Nos. 5,219,740 and 4,777,127; GB Patent No. 2,200,651; and EPPatent No. 0 345 242), alphavirus-based vectors (e.g., Sindbis virusvectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross Rivervirus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitisvirus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), andadeno-associated virus (AAV) vectors (see, e.g., PCT Publication Nos. WO94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO95/00655). Administration of DNA linked to killed adenovirus asdescribed in Curiel, Hum. Gene Ther. (1992) 3:147 can also be employed.

[0306] Non-viral delivery vehicles and methods can also be employed,including, but not limited to, polycationic condensed DNA linked orunlinked to killed adenovirus alone (see, e.g., Curiel, Hum. Gene Ther.(1992) 3:147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989)264:16985); eukaryotic cell delivery vehicles cells (see, e.g., U.S.Pat. No. 5,814,482; PCT Publication Nos. WO 95/07994; WO 96/17072; WO95/30763; and WO 97/42338) and nucleic charge neutralization or fusionwith cell membranes. Naked DNA can also be employed. Exemplary naked DNAintroduction methods are described in PCT Publication No. WO 90/11092and U.S. Pat. No. 5,580,859. Liposomes that can act as gene deliveryvehicles are described in U.S. Pat. No. 5,422,120; PCT Publication Nos.WO 95/13796; WO 94/23697; WO 91/14445; and EP 0524968. Additionalapproaches are described in Philip, Mol. Cell Biol. (1994) 14:2411, andin Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.

[0307] Identification of Anti-NGF Antagonist Antibodies

[0308] Anti-NGF antagonist antibodies can be identified or characterizedusing methods known in the art, whereby reduction, amelioration, orneutralization of an NGF biological activity is detected and/ormeasured. For example, a kinase receptor activation (KIRA) assaydescribed in U.S. Pat. Nos. 5,766,863 and 5,891,650, can be used toidentify anti-NGF agents. This ELISA-type assay is suitable forqualitative or quantitative measurement of kinase activation bymeasuring the autophosphorylation of the kinase domain of a receptorprotein tyrosine kinase (hereinafter “rPTK”), e.g. TrkA receptor, aswell as for identification and characterization of potential antagonistsof a selected rPTK, e.g., TrkA. The first stage of the assay involvesphosphorylation of the kinase domain of a kinase receptor, for example,a TrkA receptor, wherein the receptor is present in the cell membrane ofan eukaryotic cell. The receptor may be an endogenous receptor ornucleic acid encoding the receptor, or a receptor construct, may betransformed into the cell. Typically, a first solid phase (e.g., a wellof a first assay plate) is coated with a substantially homogeneouspopulation of such cells (usually a mammalian cell line) so that thecells adhere to the solid phase. Often, the cells are adherent andthereby adhere naturally to the first solid phase. If a “receptorconstruct” is used, it usually comprises a fusion of a kinase receptorand a flag polypeptide. The flag polypeptide is recognized by thecapture agent, often a capture antibody, in the ELISA part of the assay.An analyte, such as a candidate anti-NGF antagonist antibody is thenadded together with NGF to the wells having the adherent cells, suchthat the tyrosine kinase receptor (e.g. TrkA receptor) is exposed to (orcontacted with) NGF and the analyte. This assay enables identificationof antibodies that inhibit activation of TrkA by its ligand NGF.Following exposure to NGF and the analyte, the adhering cells aresolubilized using a lysis buffer (which has a solubilizing detergenttherein) and gentle agitation, thereby releasing cell lysate which canbe subjected to the ELISA part of the assay directly, without the needfor concentration or clarification of the cell lysate.

[0309] The cell lysate thus prepared is then ready to be subjected tothe ELISA stage of the assay. As a first step in the ELISA stage, asecond solid phase (usually a well of an ELISA microtiter plate) iscoated with a capture agent (often a capture antibody) which bindsspecifically to the tyrosine kinase receptor, or, in the case of areceptor construct, to the flag polypeptide. Coating of the second solidphase is carried out so that the capture agent adheres to the secondsolid phase. The capture agent is generally a monoclonal antibody, but,as is described in the examples herein, polyclonal antibodies may alsobe used. The cell lysate obtained is then exposed to, or contacted with,the adhering capture agent so that the receptor or receptor constructadheres to (or is captured in) the second solid phase. A washing step isthen carried out, so as to remove unbound cell lysate, leaving thecaptured receptor or receptor construct. The adhering or capturedreceptor or receptor construct is then exposed to, or contacted with, ananti-phosphotyrosine antibody which identifies phosphorylated tyrosineresidues in the tyrosine kinase receptor. In one embodiment, theanti-phosphotyrosine antibody is conjugated (directly or indirectly) toan enzyme which catalyses a color change of a non-radioactive colorreagent. Accordingly, phosphorylation of the receptor can be measured bya subsequent color change of the reagent. The enzyme can be bound to theanti-phosphotyrosine antibody directly, or a conjugating molecule (e.g.,biotin) can be conjugated to the anti-phosphotyrosine antibody and theenzyme can be subsequently bound to the anti-phosphotyrosine antibodyvia the conjugating molecule. Finally, binding of theanti-phosphotyrosine antibody to the captured receptor or receptorconstruct is measured, e.g., by a color change in the color reagent.

[0310] The anti-NGF antagonist antibody can also be identified byincubating a candidate agent with NGF and monitoring any one or more ofthe following characteristics: (a) binding to NGF and inhibiting NGFbiological activity or downstream pathways mediated by NGF signalingfunction; (b) inhibiting, blocking or decreasing NGF receptor activation(including TrkA dimerization and/or autophosphorylation); (c) increasingclearance of NGF; (d) treating or preventing any aspect of rheumatoidarthritis pain or osteoarthritis pain; (e) inhibiting (reducing) NGFsynthesis, production or release. In some embodiments, an anti-NGFantagonist antibody is identified by incubating an candidate agent withNGF and monitoring binding and/or attendant reduction or neutralizationof a biological activity of NGF. The binding assay may be performed withpurified NGF polypeptide(s), or with cells naturally expressing, ortransfected to express, NGF polypeptide(s). In one embodiment, thebinding assay is a competitive binding assay, where the ability of acandidate antibody to compete with a known anti-NGF antagonist for NGFbinding is evaluated. The assay may be performed in various formats,including the ELISA format. In other embodiments, an anti-NGF antagonistantibody is identified by incubating a candidate agent with NGF andmonitoring binding and attendant inhibition of trkA receptordimerization and/or autophosphotylation.

[0311] Following initial identification, the activity of a candidateanti-NGF antagonist antibody can be further confirmed and refined bybioassays, known to test the targeted biological activities.Alternatively, bioassays can be used to screen candidates directly. Forexample, NGF promotes a number of morphologically recognizable changesin responsive cells. These include, but are not limited to, promotingthe differentiation of PC12 cells and enhancing the growth of neuritesfrom these cells (Greene et al., Proc Natl Acad Sci USA. 73(7):2424-8,1976), promoting neurite outgrowth from explants of responsive sensoryand sympathetic ganglia (Levi-Montalcini, R. and Angeletti, P. Nervegrowth factor. Physiol. Rev. 48:534-569, 1968) and promoting thesurvival of NGF dependent neurons such as embryonic dorsal rootganglion, trigeminal ganglion, or sympathetic ganglion neurons (e.g.,Chun & Patterson, Dev. Biol. 75:705-711, (1977); Buchman & Davies,Development 118:989-1001 (1993). Thus, the assay for inhibition of NGFbiological activity entail culturing NGF responsive cells with NGF plusan analyte, such as a candidate anti-NGF antagonist antibody. After anappropriate time the cell response will be assayed (celldifferentiation, neurite outgrowth or cell survival).

[0312] The ability of a candidate anti-NGF antagonist antibody to blockor neutralize a biological activity of NGF can also be assessed bymonitoring the ability of the candidate agent to inhibit NGF mediatedsurvival in the embryonic rat dorsal root ganglia survival bioassay asdescribed in Hongo et al., Hybridoma 19:215-227 (2000).

[0313] Administration of an Anti-NGF Antagonist Antibody

[0314] The anti-NGF antagonist antibody can be administered to anindividual (for rheumatoid arthritis and osteoarthritis) via anysuitable route. It should be apparent to a person skilled in the artthat the examples described herein are not intended to be limiting butto be illustrative of the techniques available. Accordingly, in someembodiments, the anti-NGF antagonist antibody is administered to aindividual in accord with known methods, such as intravenousadministration, e.g., as a bolus or by continuous infusion over a periodof time, by intramuscular, intraperitoneal, intracerebrospinal,subcutaneous, intra-articular, sublingually, intrasynovial, viainsufflation, intrathecal, oral, inhalation or topical routes.Administration can be systemic, e.g., intravenous administration, orlocalized. Commercially available nebulizers for liquid formulations,including jet nebulizers and ultrasonic nebulizers are useful foradministration. Liquid formulations can be directly nebulized andlyophilized powder can be nebulized after reconstitution. Alternatively,anti-NGF antagonist antibody can be aerosolized using a fluorocarbonformulation and a metered dose inhaler, or inhaled as a lyophilized andmilled powder.

[0315] In one embodiment, an anti-NGF antagonist antibody isadministered via site-specific or targeted local delivery techniques.Examples of site-specific or targeted local delivery techniques includevarious implantable depot sources of the anti-NGF antagonist antibody orlocal delivery catheters, such as infusion catheters, an indwellingcatheter, or a needle catheter, synthetic grafts, adventitial wraps,shunts and stents or other implantable devices, site specific carriers,direct injection, or direct application. See, e.g., PCT Publication No.WO 00/53211 and U.S. Pat. No. 5,981,568.

[0316] Various formulations of an anti-NGF antagonist antibody may beused for administration. In some embodiments, the anti-NGF antagonistantibody may be administered neat. In some embodiments, anti-NGFantagonist antibody and a pharmaceutically acceptable excipient may bein various formulations. Pharmaceutically acceptable excipients areknown in the art, and are relatively inert substances that facilitateadministration of a pharmacologically effective substance. For example,an excipient can give form or consistency, or act as a diluent. Suitableexcipients include but are not limited to stabilizing agents, wettingand emulsifying agents, salts for varying osmolarity, encapsulatingagents, buffers, and skin penetration enhancers. Excipients as well asformulations for parenteral and nonparenteral drug delivery are setforth in Remington, The Science and Practice of Pharmacy 20th Ed. MackPublishing (2000).

[0317] In some embodiments, these agents are formulated foradministration by injection (e.g., intraperitoneally, intravenously,subcutaneously, intramuscularly, etc.). Accordingly, these agents can becombined with pharmaceutically acceptable vehicles such as saline,Ringer's solution, dextrose solution, and the like. The particulardosage regimen, i.e., dose, timing and repetition, will depend on theparticular individual and that individual's medical history.

[0318] An anti-NGF antibody can be administered using any suitablemethod, including by injection (e.g., intraperitoneally, intravenously,subcutaneously, intramuscularly, etc.). Anti-NGF antibodies can also beadministered via inhalation, as described herein. Generally, foradministration of anti-NGF antibodies, an initial candidate dosage canbe about 2 mg/kg. For the purpose of the present invention, a typicaldaily dosage might range from about any of 1 μg/kg to 3 μg/kg to 30μg/kg to 300 μg/kg to 3 mg/kg, to 30 mg/kg to 100 mg/kg or more,depending on the factors mentioned above. For example, an anti-NGFantibody may be administered at about 1 μg/kg, about 10 μg/kg, about 20μg/kg, about 50 μg/kg, about 100 μg/kg, about 200 μg/kg, about 500μg/kg, about 1 mg/kg, or about 2 mg/kg. For repeated administrationsover several days or longer, depending on the condition, the treatmentis sustained until a desired suppression of symptoms occurs or untilsufficient therapeutic levels are achieved to reduce pain. An exemplarydosing regimen comprises administering an initial dose of about 2 mg/kg,followed by a weekly maintenance dose of about 1 mg/kg of the anti-NGFantibody, or followed by a maintenance dose of about 1 mg/kg every otherweek. However, other dosage regimens may be useful, depending on thepattern of pharmacokinetic decay that the practitioner wishes toachieve. For example, in some embodiments, dosing from one-four times aweek is contemplated. The progress of this therapy is easily monitoredby conventional techniques and assays. The dosing regimen (including theNGF antagonist(s) used) can vary over time.

[0319] For the purpose of the present invention, the appropriate dosageof an anti-NGF antagonist antibody will depend on the anti-NGFantagonist antibody (or compositions thereof) employed, the type andseverity of the pain to be treated, whether the agent is administeredfor preventive or therapeutic purposes, previous therapy, the patient'sclinical history and response to the agent, and the discretion of theattending physician. Typically the clinician will administer an anti-NGFantagonist antibody, until a dosage is reached that achieves the desiredresult. Dose and/or frequency can vary over course of treatment.

[0320] Empirical considerations, such as the half-life, generally willcontribute to the determination of the dosage. For example, antibodiesthat are compatible with the human immune system, such as humanizedantibodies or fully human antibodies, may be used to prolong half-lifeof the antibody and to prevent the antibody being attacked by the host'simmune system. Frequency of administration may be determined andadjusted over the course of therapy, and is generally, but notnecessarily, based on treatment and/or suppression and/or ameliorationand/or delay of pain. Alternatively, sustained continuous releaseformulations of anti-NGF antagonist antibodies may be appropriate.Various formulations and devices for achieving sustained release areknown in the art.

[0321] In one embodiment, dosages for an anti-NGF antagonist antibodymay be determined empirically in individuals who have been given one ormore administration(s) of an anti-NGF antagonist antibody. Individualsare given incremental dosages of an anti-NGF antagonist antibody. Toassess efficacy of an anti-NGF antagonist antibody, an indicator of paincan be followed.

[0322] Administration of an anti-NGF antagonist antibody in accordancewith the method in the present invention can be continuous orintermittent, depending, for example, upon the recipient's physiologicalcondition, whether the purpose of the administration is therapeutic orprophylactic, and other factors known to skilled practitioners. Theadministration of an anti-NGF antagonist antibody may be essentiallycontinuous over a preselected period of time or may be in a series ofspaced dose, e.g., either before, during, or after developing pain;before; during; before and after; during and after; before and during;or before, during, and after developing pain.

[0323] In some embodiments, more than one anti-NGF antagonist antibodymay be present. At least one, at least two, at least three, at leastfour, at least five different, or more anti-NGF antagonist antibody canbe present. Generally, those anti-NGF antagonist antibodies havecomplementary activities that do not adversely affect each other.

[0324] Therapeutic formulations of the anti-NGF antagonist antibody usedin accordance with the present invention are prepared for storage bymixing an antibody having the desired degree of purity with optionalpharmaceutically acceptable carriers, excipients or stabilizers(Remington, The Science and Practice of Pharmacy 20th Ed. MackPublishing (2000)), in the form of lyophilized formulations or aqueoussolutions. Acceptable carriers, excipients, or stabilizers are nontoxicto recipients at the dosages and concentrations employed, and maycomprise buffers such as phosphate, citrate, and other organic acids;salts such as sodium chloride; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens, such asmethyl or propyl paraben; catechol; resorcinol; cyclohexanol;3-pentanol; and m-cresol); low molecular weight (less than about 10residues) polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosacchandes, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

[0325] Liposomes containing the anti-NGF antagonist antibody areprepared by methods known in the art, such as described in Epstein, etal., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang, et al., Proc.Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and4,544,545. Liposomes with enhanced circulation time are disclosed inU.S. Pat. No. 5,013,556. Particularly useful liposomes can be generatedby the reverse phase evaporation method with a lipid compositioncomprising phosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter.

[0326] The active ingredients may also be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing(2000).

[0327] Sustained-release preparations may be prepared. Suitable examplesof sustained-release preparations include semipermeable matrices ofsolid hydrophobic polymers containing the antibody, which matrices arein the form of shaped articles, e.g. films, or microcapsules. Examplesof sustained-release matrices include polyesters, hydrogels (forexample, poly(2-hydroxyethyl-methacrylate), or ‘poly(v nylalcohol)),polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acidand 7 ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), sucrose acetate isobutyrate, andpoly-D-(−)-3-hydroxybutyric acid.

[0328] The formulations to be used for in vivo administration must besterile. This is readily accomplished by, for example, filtrationthrough sterile filtration membranes. Therapeutic anti-NGF antagonistantibody compositions are generally placed into a container having asterile access port, for example, an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle.

[0329] The compositions according to the present invention may be inunit dosage forms such as tablets, pills, capsules, powders, granules,solutions or suspensions, or suppositories, for oral, parenteral orrectal administration, or administration by inhalation or insufflation.

[0330] For preparing solid compositions such as tablets, the principalactive ingredient is mixed with a pharmaceutical carrier, e.g.conventional tableting ingredients such as corn starch, lactose,sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalciumphosphate or gums, and other pharmaceutical diluents, e.g. water, toform a solid preformulation composition containing a homogeneous mixtureof a compound of the present invention, or a non-toxic pharmaceuticallyacceptable salt thereof. When referring to these preformulationcompositions as homogeneous, it is meant that the active ingredient isdispersed evenly throughout the composition so that the composition maybe readily subdivided into equally effective unit dosage forms such astablets, pills and capsules. This solid preformulation composition isthen subdivided into unit dosage forms of the type described abovecontaining from 0.1 to about 500 mg of the active ingredient of thepresent invention. The tablets or pills of the novel composition can becoated or otherwise compounded to provide a dosage form affording theadvantage of prolonged action. For example, the tablet or pill cancomprise an inner dosage and an outer dosage component, the latter beingin the form of an envelope over the former. The two components can beseparated by an enteric layer that serves to resist disintegration inthe stomach and permits the inner component to pass intact into theduodenum or to be delayed in release. A variety of materials can be usedfor such enteric layers or coatings, such materials including a numberof polymeric acids and mixtures of polymeric acids with such materialsas shellac, cetyl alcohol and cellulose acetate.

[0331] Suitable surface-active agents include, in particular, non-ionicagents, such as polyoxyethylenesorbitans (e.g. Tween™ 20, 40, 60, 80 or85) and other sorbitans (e.g. Span™ 20, 40, 60, 80 or 85). Compositionswith a surface-active agent will conveniently comprise between 0.05 and5% surface-active agent, and can be between 0.1 and 2.5%. It will beappreciated that other ingredients may be added, for example mannitol orother pharmaceutically acceptable vehicles, if necessary.

[0332] Suitable emulsions may be prepared using commercially availablefat emulsions, such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™and Lipiphysan™. The active ingredient may be either dissolved in apre-mixed emulsion composition or alternatively it may be dissolved inan oil (e.g. soybean oil, safflower oil, cottonseed oil, sesame oil,corn oil or almond oil) and an emulsion formed upon mixing with aphospholipid (e.g. egg phospholipids, soybean phospholipids or soybeanlecithin) and water. It will be appreciated that other ingredients maybe added, for example gylcerol or glucose, to adjust the tonicity of theemulsion. Suitable emulsions will typically contain up to 20% oil, forexample, between 5 and 20%. The fat emulsion can comprise fat dropletsbetween 0.1 and 1.0 μm, particularly 0.1 and 0.5 μm, and have a pH inthe range of 5.5 to 8.0.

[0333] The emulsion compositions can be those prepared by mixing a nervegrowth factor antibody with Intralipid™. or the components thereof(soybean oil, egg phospholipids, glycerol and water).

[0334] Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as set outabove. In some embodiments, the compositions are administered by theoral or nasal respiratory route for local or systemic effect.Compositions in preferably sterile pharmaceutically acceptable solventsmay be nebulised by use of gases. Nebulised solutions may be breatheddirectly from the nebulising device or the nebulising device may beattached to a face mask, tent or intermittent positive pressurebreathing machine. Solution, suspension or powder compositions may beadministered, preferably orally or nasally, from devices which deliverthe formulation in an appropriate manner.

[0335] Treatment efficacy can be assessed by methods well-known in theart.

[0336] Kits Comprising Antibodies and Polynucleotides of the Invention

[0337] The invention also provides kits comprising antibodies orpolypeptides for use in detection and/or therapy. Accordingly, in someembodiments, the kits comprise an antibody E3. In some embodiments, thekit comprises any antibody or polypeptide described herein.

[0338] In other aspects, the kits may be used for any of the methodsdescribed herein, including, for example, to treat an individual withpain (including post-surgical pain, rheumatoid arthritis pain, andosteoarthritis pain). The kits of this invention are in suitablepackaging, and may optionally provide additional components such as,buffers and instructions for use of the antibody in any of the methodsdescribed herein. In some embodiments, the kits include instructions fortreating pain. In some embodiments, the kit comprises an anti-NGFantagonist antibody described herein and instructions for treatingand/or preventing rheumatoid arthritis pain in an individual. In otherembodiments, the kit comprises an anti-NGF antagonist antibody describedherein and instructions for treating and/or preventing osteoarthritispain in an individual. In some of the embodiments, the anti-NGFantagonist antibody is antibody E3.

[0339] In another aspect, the invention provides kits comprising apolynucleotide encoding an E3 polypeptide as described herein. In someembodiments, the kits further comprise instructions for use of thepolynucleotide in any of the methods described herein.

[0340] Methods for Adjusting the Affinity of an Antibody and Methods ForCharacterizing a CDR

[0341] We have developed a novel method for characterizing a CDR of anantibody and/or altering (such as improving) the binding affinity of apolypeptide, such as an antibody, termed “library scanning mutagenesis”.Generally, library scanning mutagenesis works as follows. One or moreamino acid positions in the CDR are replaced with two or more (such as3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20)amino acids using art recognized methods. This generates small librariesof clones (in some embodiments, one for every amino acid position thatis analyzed), each with a complexity of two or more members (if two ormore amino acids are substituted at every position). Generally, thelibrary also includes a clone comprising the native (unsubstituted)amino acid. A small number of clones, e.g., about 20-80 clones(depending on the complexity of the library), from each library arescreened for binding affinity to the target polypeptide, and candidateswith increased, the same, decreased or no binding are identified.Methods for determining binding affinity are well-known in the art. Insome embodiments, binding affinity is determined using BIAcore surfaceplasmon resonance analysis, which detects differences in bindingaffinity of about 2-fold or greater. BIAcore is particularly useful whenthe starting antibody already binds with a relatively high affinity, forexample a K_(D) of about 10 nM or lower. Screening using BIAcore surfaceplasmon resonance is described in the Examples, herein.

[0342] In other embodiments, binding affinity is determined using KinexaBiocensor, scintillation proximity assays, ELISA, ORIGEN immunoassay(IGEN), fluorescence quenching, fluorescence transfer, and/or yeastdisplay. In other embodiments, binding affinity is screened using asuitable bioassay.

[0343] In some embodiments, every amino acid position in a CDR isreplaced (in some embodiments, one at a time) with all 20 natural aminoacids using art recognized mutagenesis methods (some of which aredescribed herein). This generates small libraries of clones (in someembodiments, one for every amino acid position that is analyzed), eachwith a complexity of 20 members (if all 20 amino acids are substitutedat every position).

[0344] In some embodiments, the library to be screened comprisessubstitutions in two or more positions, which may be in the same CDR orin two or more CDRs. Thus, in some embodiments, the library comprisessubstitutions in two or more positions in one CDR. In other embodiments,the library comprises substitution in two or more positions in two ormore CDRs. In still other embodiments, the library comprisessubstitution in 3, 4, 5, or more positions, said positions found in two,three, four, five or six CDRs. In some embodiments, the substitution isprepared using low redundancy codons. See, e.g., Table 2 of Balint etal., (1993) Gene 137(1):109-18).

[0345] In some embodiments, the CDR is CDRH3 and/or CDRL3. In otherembodiments, the CDR is one or more of CDRL1, CDRL2, CDRL3, CDRH1,CDRH2, and/or CDRH3. In some embodiments, the CDR is a Kabat CDR, aChothia CDR, or an extended CDR.

[0346] Candidates with improved binding may be sequenced, therebyidentifying a CDR substitution mutant which results in improved affinity(also termed an “improved” substitution). For example, as demonstratedin Example 1, use of this method permitted identification of a singlesubstitution which improved binding, even when an estimated 18 othersubstitutions at the same amino acid position resulted in no binding(i.e., loss of antibody function). Candidates that bind may also besequenced, thereby identifying a CDR substitution which retains binding.

[0347] In some embodiments, multiple rounds of screening are conducted.For example, candidates (each comprising an amino acid substitution atone or more position of one or more CDR) with improved binding are alsouseful for the design of a second library containing at least theoriginal and substituted amino acid at each improved CDR position (i.e.,amino acid position in the CDR at which a substitution mutant showedimproved binding). Preparation, and screening or selection of thislibrary is discussed further below.

[0348] Library scanning mutagenesis also provides a means forcharacterizing a CDR, in so far as the frequency of clones with improvedbinding, the same binding, decreased binding or no binding also provideinformation relating to the importance of each amino acid position forthe stability of the antibody-antigen complex. For example, if aposition of the CDR retains binding when changed to all 20 amino acids,that position is identified as a position that is unlikely to berequired for antigen binding. Conversely, if a position of CDR retainsbinding in only a small percentage of substitutions, that position isidentified as a position that is important to CDR function. Thus, thelibrary scanning mutagenesis methods generate information regardingpositions in the CDRs that can be changed to many different amino acid(including all 20 amino acids), and positions in the CDRs which cannotbe changed or which can only be changed to a few amino acids. Thisaspect is discussed and exemplified in Example 1.

[0349] In some embodiments, candidates with improved affinity arecombined in a second library, which includes the improved amino acid,the original amino acid at that position, and may further includeadditional substitutions at that position, depending on the complexityof the library that is desired, or permitted using the desired screeningor selection method. In addition, if desired, adjacent amino acidposition can be randomized to at least two or more amino acids.Randomization of adjacent amino acids may permit additionalconformational flexibility in the mutant CDR, which may in turn, permitor facilitate the introduction of a larger number of improvingmutations. In some embodiments, the library also comprises substitutionat positions that did not show improved affinity in the first round ofscreening.

[0350] The second library is screened or selected for library memberswith improved and/or altered binding affinity using any method known inthe art, including screening using BIAcore surface plasmon resonanceanalysis, and selection using any method known in the art for selection,including phage display, yeast display, and ribosome display.

[0351] Advantages of the Methods for Adjusting the Affinity of anAntibody and Characterizing a CDR

[0352] The methods are useful for pre-screening CDR amino acid positionsin order to identify amino acid substitutions that improve binding orretain binding. Pre-identification of important residues, substitutionthat improve binding and/or substitutions that retain antibody functionpermits efficient design and screening of an affinity maturationlibrary.

[0353] The present method is also useful for characterizing a CDR, andprovides comprehensive information regarding the importance of eachamino acid position in a CDR for binding to antigen. The present methodmay also be used to identify substitutions that improve binding.

[0354] The use of small libraries, in which each position may berandomized (in some embodiments, one at a time), permits screening ofsubstitution mutants using sensitive methods such as BIAcore whichprovide detailed kinetic information. Screening methods are generallyimpractical when larger libraries are screened. Instead, selectionmethods, such as phage display, yeast display, and ribosome display, arecommonly used to identify clones that retain binding. Phage display andELISA assays may depend heavily on the concentration of the proteinsample prepared from the clone, and thus tend to be heavily biasedtowards clones that have increased expression, increased stability, ordecreased toxicity, rather than identifying clones with increasedbinding affinity. In addition, differences in expression level of theclones may mask small improvements in binding affinity. Thesedisadvantages are particularly acute when an antibody with high bindingaffinity is used as the starting material, because very low levels ofantigen must be used in order for screening to be sufficientlystringent.

[0355] By contrast, the methods of the invention, such as randomizationat each position (in some embodiments, one position at a time), permitsintroduction and characterization of the effect of the substitution of,for example, all 20 amino acids at a given position. This analysisprovides information as to how many substitutions at a given positionare tolerated (i.e., retain antibody binding), which in turn, providesinformation relating to the importance of each amino acid for antibodyfunction. Further, substitutions that result in improved binding can beidentified, even under circumstances in which many or most of thesubstitutions at a given position yield non-functional (non-binding)antibodies. By contrast, alanine-scanning mutagenesis, which is commonlyused to identify important CDR positions, provides information relatingto whether the substitution of alanine permits or prevents binding.Generally, positions at which an alanine substitution prevents bindingare removed from the affinity maturation library. In many cases,however, alanine may be a poor substitute at the CDR position.

[0356] The present methods also permit identification andcharacterization of the effect of single CDR mutations. By contrast,methods such as phage display introduce and select many mutationssimultaneously, and thus potentially increase the risk that positivemutations will be masked by the presence of a detrimental mutationpresent in a particular clone.

[0357] The present methods are also useful for improving affinity whileretaining the binding specificity of the original (starting) antibody,insofar as the present methods permit identification of small numbers ofmutations (e.g., 1, 2, 3, 4, or 5 mutations in a single CDR) that resultin improved binding affinity. By contrast, methods such as phage displaytypically improve binding affinity using multiple mutations at once,which may result in shifting specificity of the antibody and/orincreasing undesirable cross-reactivity.

[0358] The following examples are provided to illustrate, but not tolimit, the invention.

EXAMPLES Example 1 Humanization and Affinity Maturation of MouseAntagonist Anti-NGF Antibody 911

[0359] A. General Methods

[0360] The following general methods were used in this example.

[0361] Library Generation

[0362] Libraries were generated by PCR cassette mutagenesis withdegenerate oligonucleotides as described in Kay et al. (1996), Phagedisplay of peptides and proteins: a laboratory manual, San Diego,Academic Press (see, pages pg 277-291). The doping codon NNK was used torandomize one amino acid position to include 20 possible amino acids. Torandomize one amino acid position to include only a subset of aminoacids with specific properties, doping codons were used as described inBalint et al, (1993) Gene 137(1):109-18). Site directed mutagenesis wasperformed using recombinant PCR as described in Innis et al, (1990) PCRprotocols: A guide to methods and applications (see, pp. 177-183).

[0363] Small Scale Fab Preparation

[0364] Small scale expression in 96 wells plates was optimized forscreening Fab libraries. Starting from E. coli transformed with a Fablibrary, colonies were picked to inoculate both a master plate (agarLB+Ampicillin (50 μg/ml)+2% Glucose) and a working plate (2 ml/well, 96well/plate containing 1.5 mL of LB+Ampicillin (50 μg/ml)+2% Glucose).Both plates were grown at 30° C. for 8-12 hours. The master plate wasstored at 4° C. and the cells from the working plate were pelleted at5000 rpm and resuspended with 1 mL of LB+Ampicillin (50 μg/ml)+1 mM IPTGto induce expression of Fabs. Cells were harvested by centrifugationafter 5 h expression time at 30° C., then resuspended in 500 μL ofbuffer HBS-EP (100 mM HEPES buffer pH 7.4, 150 mM NaCl, 0.005% P20, 3 mMEDTA). Lysis of HBS-EP resuspended cells was attained by one cycle offreezing (−80° C.) then thawing at 37° C. Cell lysates were centrifugedat 5000 rpm for 30 min to separate cell debris from supernatantscontaining Fabs. The supernatants were then injected into the BIAcoreplasmon resonance apparatus to obtain affinity information for each Fab.Clones expressing Fabs were rescued from the master plate to sequencethe DNA and for large scale Fab production and detailed characterizationas described below.

[0365] Large Scale Fab Preparation

[0366] To obtain detailed kinetic parameters, Fabs were expressed andpurified from large cultures. Erlenmeyer flasks containing 200 mL ofLB+Ampicillin (50 μg/ml)+2% Glucose were inoculated with 5 mL of overnight culture from a selected Fab-expressing E. coli clone. Clones wereincubated at 30° C. until an OD₅₅₀ nm of 1.0 was attained and theninduced by replacing the media for 200 ml, of LB+Ampicillin (50 μg/ml)+1mM IPTG. After 5 h expression time at 30° C., cells were pelleted bycentrifugation, then resuspended in 10 mL PBS (pH 8). Lysis of the cellswas obtained by two cycles of freeze/thaw (at −80° C. and 37° C.,respectively). Supernatant of the cell lysates were loaded onto Ni-NTAsuperflow sepharose (Qiagen, Valencia. CA) columns equilibrated withPBS, pH 8, then washed with 5 column volumes of PBS, pH 8. IndividualFabs eluted in different fractions with PBS (pH 8)+300 mM Imidazol.Fractions containing Fabs were pooled and dialized in PBS, thenquantified by ELISA prior to affinity characterization.

[0367] Full Antibody Preparation

[0368] For expression of full antibodies, heavy and light chain variableregions were cloned in 2 mammalian expression vectors (Eb.911.E3 orEb.pur.911.3E for light chain and Db.911.3E for heavy chain; describedherein) and transfected using lipofectemine into HEK 293 cells fortransient expression. Antibodies were purified using protein A usingstandard methods.

[0369] Biacore Assay

[0370] Affinities of anti-NGF Fabs and monoclonal antibodies weredetermined using the BIAcore3000™ surface plasmon resonance (SPR) system(BIAcore, INC, Piscaway N.J.). CM5 chips were activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiinide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Human NGF was diluted into 10 mM sodium acetate pH 4.0 and injected overthe activated chip at a concentration of 0.005 mg/mL. Using variableflow time across the individual chip channels, two ranges of antigendensity were achieved: 100-200 response units (RU) for detailed kineticstudies and 500-600 RU for screening assays. The chip was blocked withethanolamine. Regeneration studies showed that a mixture of Pierceelution buffer (Product No. 21004, Pierce Biotechnology, Rockford, Ill.)and 4 M NaCl (2:1) effectively removed the bound Fab while keeping theactivity of hNGF on the chip for over 200 injections. HBS-EP buffer(0.01M HEPES, pH 7.4, 0.15 NaCl, 3 mM EDTA, 0.005% Surfactant P29) wasused as running buffer for all the BIAcore assays.

[0371] Screening Assay

[0372] A screening BIAcore assay was optimized to determine the affinityof Fab clones from libraries. Supernatants of small culture lysates wereinjected at 50 μl/min for 2 min. Dissociation times of 10 to 15 minuteswere used for determination of a single exponential dissociation rate(k_(off)) using BIAevaluation software. Samples that showed k_(off)rates in the same range as the template used to create the library(clone 8L2-6D5, k_(off) 1×10⁻³ s⁻¹) were injected for confirmation anddissociation times of up to 45 min were allowed to obtain better k_(off)values. Clones showing improved (slower) k_(off) values were expressedat large scale and full kinetic parameters, k_(on) and k_(off), weredetermined on purified protein. The assay was capable of detectingdifferences in affinity that were approximately 2-fold or larger.

[0373] Affinity Determination Assay

[0374] Serial dilutions (0.1-10×estimated K_(D)) of purified Fab sampleswere injected for 1 min at 100 μL/min and dissociation times of up to 2h were allowed. The concentrations of the Fab proteins were determinedby ELISA and/or SDS-PAGE electrophoresis using as a standard a Fab ofknown concentration (as determined by amino acid analysis). Kineticassociation rates (k_(on)) and dissociation rates (k_(off)) wereobtained simultaneously by fitting the data to a 1:1 Langmuir bindingmodel (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B. (1994). MethodsEnzymology 6. 99-110) using the BIAevaluation program. Equilibriumdissociation constant (K_(D)) values were calculated as k_(off)/k_(on).

[0375] B. Humanization and Affinity Maturation of Mouse AntagonistAnti-NGF Antibody 911

[0376] The mouse antagonist anti-NGF antibody, 911 (see Hongo et al,(2000) Hybridoma 19(3):215-227) was selected for humanization andaffinity maturation. Mab 911 binds human and rat NGF with high affinityand exhibits no significant cross-reactivity with the neurotrophins NT3,NT4/5 or BDNF. See Hongo, id. The affinity of the papain-cleaved Fabfragment of mouse Mab 911 was determined using BIAcore analysis asdescribed above. The papain-cleaved Fab fragment of mouse Mab 911 boundhuman NGF with a K_(D) of approximately 10 nM.

[0377] Humanization and affinity maturation was conducted in severalsteps, as follows:

[0378] (1) Preparation of CDR-grafted template. The light chain extendedCDRs of antibody 911 (i.e., including both the Kabat and Chothia CDRregions) were grafted into the human gemmline acceptor sequences 08 withJK2 and the heavy chain extended CDRs of antibody 911 were grafted in tohuman germline acceptor sequence VH4-59 with JH4. The amino acidsequences of the human germline acceptor sequences are shown in FIGS. 1Aand 1B. Amino acid numbering is sequential. Using the protein frameworksnoted above, DNA sequences were designed for synthetic genes encodinghuman framework with the murine CDRs. These humanized heavy and lightvariable domains were termed hVH and hVL respectively. Codons wereoptimized for E. coli and hamster usage. Several overlappingoligonucleotides (69-90 bases in length) extending the full length ofthe hVL and hVH with two short flanking primers for each chain were usedto separately synthesize the two genes by recursive PCR essentially asdescribed in Prodromou et al, (1992) Protein Eng 5(8): 827-9. ResultingDNA fragments of the correct length were gel purified and then clonedinto an E. coli bicistronic expression plasmid (ampicillin resistant).Expression of the antibodies was under control of an IPTG inducible lacZpromoter similar to that described in Barbas (2001) Phage display: alaboratory manual, Cold Spring Harbor, N.Y., Cold Spring HarborLaboratory Press (see Vector pComb3X, at pg 2.10), however,modifications included addition and expression of the followingadditional domains: the human Kappa light chain constant domain (seeGenBank Accession No. CAA09181) and the CH1 constant domain of IgG2ahuman immunoglobulin (GenBank Accession No. P01859).

[0379] The amino acid sequences of the variable regions of theCDR-grafted antibody (also termed the “template”), termed 8L2-4D5, arealso shown in FIGS. 1A and 1B. The affinity of 8L2-4D5 was determinedusing BIAcore analysis as described above. 8L2-4D5 bound human NGF witha K_(D) of approximately 38 nM.

[0380] (2) Introduction of a point mutation into the framework sequence.The V71K substitution was introduced into the CDR-grafted heavy chainusing recombinant PCR site directed mutagenesis as described in Innis etal, (1995) PCR strategies, San Diego, Academic Press. This substitutionreplaced the human framework residue with the corresponding mouseframework residue. The resulting antibody was termed 8L2-6D5, and theamino acid sequence of the heavy chain variable region of 8L2-6D5 isshown in FIG. 1A. The affinity of 8L2-6D5 was determined using BIAcoreanalysis as described above. The Fab fragment of 8L2-6D5 bound human NGFwith a Kd of approximately 15 nM. 8L2-6D5 was chosen as template foraffinity maturation.

[0381] (3) Humanization and affinity maturation of CDRs L1, L2, H1 andH2. CDRs L1, L2, H1 and H2 were subjected to humanization and affinitymaturation. Amino acid positions in CDRs L1, L2, Hi, and H2 wereidentified that are not essential for the structure of the CDRs based onthe Chothia canonical structure (see Al-Lazikani et al (1997) J. Mol.Biol. 273(4):927-48); and subjected to randomization as follows. Twolibraries were prepared containing the light chain mutations or heavychain mutations shown in Table 2, and the grafted (mouse) CDR L3 or CDRH3, respectively, using PCR cassette mutagenesis with degenerateoligonucleotides as described in Kay et al. (1996), Phage display ofpeptides and proteins: a laboratory manual, San Diego, Academic Press,using doping codons as described in Balint et al, (1993) Gene137(1):109-18). Generally, the amino acid residues were altered toresidues that are more common in human antibodies, based on alignmentsof antibody 911 light chain and heavy chain amino acid sequences withhuman germline antibody sequences. The wildtype (unsubstituted) aminoacid residue was also represented in the library with the exception ofCDR H2 residue 50, a methionine, in which the wildtype methionine wasnot represented in the library. Methionine residues are subject tooxidation; thus, replacement of that residue was expected to improvestability of the resulting antibody. The libraries of Fabs were clonedinto vector pComb3X plus the human CH1 and CK regions, as describedabove. TABLE 2 1. Heavy chain H1/H2 library: CDR-H1 134 was changed toF, L, V, S, P, T, A, or I N35 was changed to N, T, S, or Y CDR-H2 M50was changed to all 20 natural amino acids A62 was changed to A or S L63was changed to L or V 2. Light chain L1/L2 library CDR-L1 S26 waschanged to S, A, V, or F D28 was changed to D, A, S, or Y H32 waschanged to H, N, K, D, E, Q, or Y CDR-L2 Y50 was changed to Y, D, A, orS I51 was changed to I, T, A, or V F54 was changed to F or L S56 waschanged to S and T

[0382] For affinity screening experiments, each library was furtherpaired with the corresponding CDR-grafted light or heavy chain (forexample, the H1/H2 library was paired with CDR-grafted light chain), theantibody was expressed, and affinity to human NGF of the individualclones was screened using the BIACORE surface plasmon resonance (SPR)system (BIAcore, Inc. Piscataway, N.J.) according to the manufacturer'sinstructions and as described above. k_(off), k_(on), and K_(D) weredetermined. Antibody clones were ranked based on k_(off) rates, sincegenerally most variation in affinity is seen in k_(off) rates, andfurther because k_(off) rates are independent of antibody concentration.

[0383] The sequence of clones that bound was determined and the sequenceof clones that bound is shown in table 3. TABLE 3 L1 and L2 amino acidsequences, H1 and H2 amino acid sequences, and kinetic data for clonesthat bound following affinity screening of H1/H2 or L1/L2 libraryclones. CDR 1-2 mutants kinetic data Light chain library clones Pairedwith 8L2 CDRL1 CDRL2 k_(off) *K_(D) heavy chain AA sequence AA sequence(s − 1) (nM) 8L2-6D5 RASQDISNHLN YISRFHS **1e − 3 25 (control) (SEQ IDNO:12) (SEQ ID NO:13) L129 RASQSISNNLN YTSRFHS  4.5e − 4 11 (SEQ IDNO:18) (SEQ ID NO:19) L208 RASQYISNHLN YTSRFHS  4.6e − 4 11 (SEQ IDNO:20) (SEQ ID NO:21) L97 RASQSISNQLN YVSRFHS  5.6e − 4 14 (SEQ IDNO:22) (SEQ ID NO:23) L81 RAFQAISNQLN YISRFHT  7.4e − 4 18 (SEQ IDNO:24) (SEQ ID NO:25) L6 RAFQSISNQLN YASRFHS  8.2e − 4 20 (SEQ ID NO:26)(SEQ ID NO:27) Heavy chain library clones Paired with 6D5 CDRH1 CDRH2k_(off) *K_(D) Light chain AA sequence AA sequence (s − 1) (nM) 8L2-6D5GFSLIGYDIN MIWGDGTTDYNSAL   1e − 3 25 (control) (SEQ ID NO:9) (SEQ IDNO:10) H109 GFSLIGYDSN IIWGDGTTDYNSAL  1.6e − 4 4 (SEQ ID NO:28) (SEQ IDNO:29) H19 GFSLIGYDLN IIWGDGTTDYNSAV  2.4e − 4 6 (SEQ ID NO:30) (SEQ IDNO:31) H222 GFSLIGYDVT GIWGDGTTDYNSAV  3.8e − 4 9.5 (SEQ ID NO:32) (SEQID NO:33) H225 GFSLIGYDVT GIWGDGTTDYNSSV  3.8e − 4 9.5 (SEQ ID NO:34)(SEQ ID NO:35) H18 GFSLIGYDAT GIWGDGTTDYNSAV  4.2e − 4 10.5 (SEQ IDNO:36) (SEQ ID NO:37) H9 GFSLIGYDVS IIWGDGTTDYNSSV  4.1e − 4 10.2 (SEQID NO:38) (SEQ ID NO:39) H227 GFSLIGYDIS QIWGDGTTDYNSSV  5.4e − 4 13.5(SEQ ID NO:40) (SEQ ID NO:41) H17 GFSLIGYDAS GIWGDGTTDYNSSV  6.1e − 415.2 (SEQ ID NO:42) (SEQ ID NO:43) H28 GFSLIGYDST SIWGDGTTDYNSAL  7.5e −4 18.7 (SEQ ID NO:44) (SEQ ID NO:45)

[0384] CDRs containing the following substitutions retained binding:

[0385] CDR-H1

[0386] I34: S, L, V, I and A bound.

[0387] N35: N, T and S bound.

[0388] CDR-H2

[0389] M50: M, I, G, Q, S, L bound.

[0390] A62: A and S bound.

[0391] L63: Land V bound.

[0392] CDR-L1

[0393] S26: S, and F bound.

[0394] D28: D, S, A, Y bound.

[0395] H32: H, N, Q bound.

[0396] CDR-L2

[0397] Y50: Y bound.

[0398] I51: I, T, V, A, bound.

[0399] F54: F bound

[0400] S56: S and T bound

[0401] CDRs containing the following substitutions were selectedgenerally based on binding affinity and combined into a single clone,termed H19-L129:

[0402] CDR-H1: I34L; N35N (no change)

[0403] CDR-H2: M50I; A62A (no change); L63V

[0404] CDR-L1: S26S (no change); D28S; H₃₂N

[0405] CDR-L2: Y50Y (no change); I51T; F54F (no change); S56S (nochange)

[0406] These mutations were combined (by amplifying the H and L chainsby PCR, cutting the PCR products and vector (pRN8) with restrictionenzyme and performing a 3 fragment ligation) into a single clone, termedH19-L129, which also included the grafted H3 and L3 CDRs. The sequenceof the heavy chain and light chain variable regions of H19-L129 is shownin FIGS. 1A and 1B, and Table 4 shows the amino acid sequence of CDRsL1, L2, H1 and H2. H19-L129 bound NGF with a K_(D) of approximately 1nM, as determined using BIAcore analysis as described herein. TABLE 4Amino acid sequence of CDRs H1, H2, L1 and L2 and kinetic data forcombined clone H19-L129. Combination clone: mutations CDRL1 CDRL2 inCDRs H1, H2, CDRH1 CDRH2 k_(off) *K_(D) L1, L2 AA sequence AA sequence(s − 1) (nM) H19-L129 CDR-L1: CDRL2: 1.1e − 4 3.5 RASQSISNNLN YTSRFHS(SEQ ID NO:18) (SEQ ID NO:19) CDR H1: CDR-H2: GFSLIGYDLN IIWGDGTTDYNSAV(SEQ ID NO:30) (SEQ ID NO:31)

[0407] (4) Affinity maturation of H3 and L3 CDRs. Affinity maturation ofthe H3 and L3 CDRs was carried out in two steps. First, in a processtermed “library scanning mutagenesis”, each amino acid residue in H3 andL3 was individually prescreened in order to identify amino acidpositions at which a mutation resulted in increased binding affinity tohuman NGF. Based on the results of the library scanning mutagenesis(also termed “small library randomization analysis”), a subset of aminoacid positions in H3 and L3 were selected for preparation of theaffinity maturation library, and the affinity maturation library wasscreened for affinity to human NGF using BIAcore analysis as describedherein. It is appreciated that these techniques can be generallyapplied.

[0408] (a) Library Scanning Mutagenesis

[0409] Each amino acid position in the H3 and L3 CDRs was individuallypre-screened for substitutions which resulted in increased bindingaffinity to human NGF. The frequency of amino acid substitutions at anygiven position that resulted in improved binding, the same binding,worse binding or no binding provided information relating to relating topositions in the CDRs that can be changed to many different amino acid(including all 20 amino acids), and positions in the CDRs which cannotbe changed or which can only be changed to a few amino acids. Amino acidsubstitutions resulting in increased binding affinity were alsoidentified. Based on the results of this screening, a subset of aminoacid positions in CDRs H3 and L3 were selected for preparation of anaffinity maturation library.

[0410] Individual Fab libraries were prepared in which each amino acidof L3 and H₃CDRs was randomized to all 20 amino acids, one at a time,resulting in several (5 libraries for the light chain and 13 librariesfor the heavy chain) small libraries, each with a complexity of 20 aminoacid possibilities at each amino acid position. In all cases, the native(i.e., unchanged) amino acid was represented in the library. Librarieswere prepared by PCR cassette mutagenesis with degenerateoligonucleotides as described in Kay et al. (1996), Phage display ofPeptides and Proteins: a laboratory manual, San Diego, Academic Press,using the doping codon NNK to randomize one amino acid position toinclude 20 possible amino acids. The 8L2-6D5 (the CDR grafted antibody,having the framework mutation V71K) served as the template for libraryconstruction because the lower affinity of the CDR grafted antibodypermitted easier detection of differences in affinity in H3 and L3mutants during screening. Thus, each member of a library contained aCDR3 (either H3 or L3) with one amino acid substitution, and 5 graftedCDRs.

[0411] 20-80 clones from each small library were screened using BIAcoreanalysis as described herein. Samples were simultaneously analyzed byBIAcore for binding affinity to NGF in one channel of the BIAcore chipand for presence of Fab by binding to a penta-histag antibody in anotherchannel of the sensor chip, to detect the his tag at the C terminus ofthe heavy chain. Clones that expressed protein were classified as havingthe same affinity, worse affinity, better affinity or no binding, usingk_(off) to classify: The results of this analysis are shown in Table 5.TABLE 5 Clones that expressed protein were classified as having the sameaffinity, worse affinity, better affinity or no binding, based on koff.Percentage of AAs that same retain better ≧1e−3, Worse binding mutation1e−3< 2e−3< ≧2e−3 no bind capacity Light chain L_S91X 13% 40% 20% 26%50% L_K92X 100%  ˜100%    L_T93X 93%  7% 93% L_L94X 40% 60% 40% L_Y96X13% 80%  7% 13% Heavy chain H_G98X 50% 37% 13% 50% H_G99X 46% 54% 46% HY100X 26% 73% 26% H Y101X  6% 12% 82%  6% H_Y102X  7% 25    68%  7%H_G103X  4% 21% 16% 58% 25% H_T104X 20% 30% 50% 20% H_S105X 10% 25% 26%39% 35% H_Y106X 75% 25% 75% H_Y107X  8% 46% 46%  8% H_F108X 23% 27% 50%23% H_D109X 29% 46% 25% 29% H_Y110X 90%  5%  5% 90%

[0412] The sequence of all clones with improved affinity was determined,revealing the frequency and identity of amino acid substitutions thatresulted in increased affinity. In addition, a few clones that retainedan affinity similar to the 812-6D5 clone were selected from eachlibrary, in order to ascertain amino acid sequence substitutions thatwere permitted at a given position, even though the substitution did notnecessarily increase binding affinity. The results of this analysis aresummarized in Table 6. TABLE 6 k_(off)(s − 1) K_(D)* (nM) 1E−3 25 CDR H3mutations (8L2-6D5 template, including antibody 911 CDR-H3 amino acidsequence: GGYYYGTSYYFDY (SEQ ID NO: 11) Y100L 1.2E−3 30 Y100R 1.1E−3 27Y101W 5.6E−4 14 G103A 1.6E−4 4 T104S 2.2E−3 55 S105A 5.1E−4 13 S105T6.4E−4 16 Y106R 1.6E−3 40 Y106T 2.0E−3 50 Y106M 2.7E−3 67 Y107F 1.4E−335 F108W 1.22E−3 30 D109N 1.5E−3 37 D109G 1E−3 25 Y110K 1.4E−3 35 Y110S1.5E−3 37 Y110R 1.6E−3 40 Y110T 1.7E−3 42 CDR L3 mutations (8L2-6D5template, including wildtype (unsubstituted) CDR-L3 amino acid sequence:QQSKTLPYT (SEQ ID NO: _14) S91E 2.5E−4 6 Y96R 1.7E−3 42

[0413] Several mutations resulted in increased binding affinity. Atleast the following mutations resulted in significantly increasedbinding affinity as compared with the 8L2-6D5 template: (H_Y101W (CDRsequence GGYWYGTSYYFDY (SEQ ID NO:46)); H_S105A (CDR sequenceGGYYYGTAYYFDY (SEQ ID NO:47)); H_S 105T (CDR sequence GGYYYGTTYYFDY (SEQID NO:48)); H_G103A (CDR sequence GGYYYATSYYFDY (SEQ ID NO:49); andL_S91E (CDR sequence QQEKTLPYT (SEQ ID NO:50)).

[0414] The results of this experiment were used to guide selection ofamino acid positions for generation of the affinity maturationlibraries.

[0415] This experiment also provided information regarding the frequencyof amino acid substitutions at any given position that resulted inimproved binding, the same binding, worse binding or no binding, asshown in Table 5. This information permitted identification of aminoacid positions in the CDRs that could be changed to many different aminoacid (including all 20 amino acids), and positions in the CDRs whichcould be changed to a few amino acids or a very few amino acids (in someembodiments, no amino acids). These results also demonstrated amino acidsubstitutions that increased binding affinity.

[0416] (b) Affinity Maturation

[0417] Next, the results of the small library randomization analysis(above) were used to select residues for production of the H3 and L3libraries for affinity maturation of the H3 and L3 CDRs. Residues Y10and G103 of CDR H3 and residues S91 and K92 of CDR L3 were selected forproduction of the H3 and L3 libraries for affinity maturation of the H3and L3 CDRs.

[0418] This library combined mutations in H3 and L3 at the same time inCDR-grafted clone 8L2-6D5, and separately in the background of H19-L129,and had a diversity of 80 different clones. Table 7 shows the amino acidresidues selected for substitution and the amino acids that weresubstituted at each position.

[0419] Table 7. Amino acid residues in H3 and L3 selected forsubstitution and the amino acids that were substituted at each position

[0420] CDR-H3:

[0421] Y101 was changed to Y and W, C. (Note that C was included becauseuse of codon TRS in one degenerated oligonucleotide also generated codonC).

[0422] G103 was changed to A, P, S

[0423] CDR-L3:

[0424] S91 was changed to E.

[0425] K92 was changed to all twenty amino acids. A, R, K, and H bound.

[0426] Each polypeptide was expressed as a Fab, and affinity to humanNGF of 96 individual clones was screened for each library using BIACOREanalysis according to the manufacturer's instructions and describedabove. The results of this analysis are shown in Table 8. TABLE 8 CDR L3H3 COMBINATION mutations k_(off (s − 1)) K_(D)* (nM) (8L2-6D5 template)  1E − 3 25 L_S91E; L_K92A 5.5E − 4 13 (CDR sequence QQEATLPYT (SEQ IDNO:51)) H Y101W; H_G103A (CDR sequence GGYWYATSYYFDY (SEQ ID NO:52))L_S91E; L_K92R 1.0E − 4 25 (CDR sequence QQERTLPYT (SEQ ID NO:53))H_Y101W; H_G103A (CDR sequence GGYWYATSYYFDY (SEQ ID NO:54)) CDR L3 H3COMBINATION mutations k_(off)(s − 1) K_(D)* (nM) (H19-L129 template,H1H2L1L2 matured) 1.1e − 4 L_S91E; L_K92H 1.2E − 5 0.3 (CDR sequenceQQEHTLPYT (SEQ ID NO:55)) H_Y101W; H_G103A (CDR sequence GGYWYATSYYFDY(SEQ ID NO:56)) (CLONE E3) L_S91E; L_K92S 4.7E − 5 1.1 (CDR sequenceQQESTLPYT (SEQ ID NO:57)) H_Y101W; H_G103S (CDR sequence GGYWYSTSYYFDY(SEQ ID NO:58)) L_S91E; L_K92K   2E − 5 0.5 (CDR sequence QQEKTLPYT (SEQID NO:59)) H_Y101Y; H_G103A (CDR sequence GGYYYATSYYFDY (SEQ ID NO:60))L_S91E; L_K92R 1.4E − 5 0.35 (CDR sequence QQERTLPYT (SEQ ID NO:61))H_Y101W; H_G103A (CDR sequence GGYWYATSYYFDY (SEQ ID NO:62)) (CLONE 3C)L_S91E; L_K92R 1.5E − 5 0.37 (CDR sequence QQERTLPYT (SEQ ID NO:63))H_Y101Y; H_G103A (CDR sequence GGYYYATSYYFDY (SEQ ID NO:64))

[0427] Based on binding affinity, the best clones, E3 (interchangeablytermed “3E”) and 3C, were selected for further characterization. E3comprised the following CDR substitutions: CDR-H3: Y101W, G103A; andCDR-L3: S91E, K92H, which were combined into a single clone which alsoincluded the following L1, L2, H1 and H2 mutations:

[0428] CDR-H1: I34L;

[0429] CDR-H2: M50I; L63V;

[0430] CDR-L1: D28S; H₃₂N;

[0431] CDR-L2: I51T.

[0432] The sequence of the heavy chain and light chain variable regionsof E3 is shown in FIGS. 1A and 1B. 3C comprised the following CDRsubstitutions: CDR-L3: S91E; K92R; CDRH3: Y101W; G103A, which werecombined into a single clone which also included the L1, L2, H1 and H2mutations described for clone 3E.

[0433] E and 3C sequences were cloned into mammalian expression vectorsfor production of Fab and full antibody, and expressed in HEK293 cellsand purified using Ni-NTA or protein A chromatography. Pure protein wasaccurately quantified by amino acid analysis.

[0434] The binding affinities to human NGF of Fabs E3 and 3C weremeasured using BIAcore analysis according to the manufacturer'sinstructions and as described above, except that 100 RU NGF was used onchip to prevent a rebinding effect. Briefly, several concentrations ofantibodies (Fabs) were injected for 2 minutes onto a CM5 chip with 100RU of immobilized human NGF on it, and permitted to dissociate for 1800seconds. Mouse antibody 911 (Fab) was analyzed as a control. Data wasanalyzed using BIAevaluation software following the manufacturer'sinstructions. The results of the analysis of antibody E3 and 911 areshown in FIGS. 9 and 10. E3 bound human NGF with a K_(D) ofapproximately 0.07 nM (and with a k_(on) of about 6.0e5 M-1s-1, and ak_(off) of about 4.2e-5 s-1). 3C bound human NGF with a K_(D) ofapproximately 0.35 nM (with a k_(off) of about 1.4E-5). By contrast,mouse antibody 911 bound NGF with a K_(D) of 3.7 nM, k_(off) of8.4×10⁻⁵s⁻¹ and k_(on) of 2.2×10⁴Ms⁻¹.

[0435] Antibody E3 (interchangeably termed 3E) was selected for furtheranalysis based on the high binding affinity. To test the ability of E3to prevent the interaction of NGF with the NGF receptors trkA and p75,2.5 nM of human NGF was premixed and incubated for one hour with 0 to 50nM of antibody E3 (Fab). After the incubation, samples were injected at10 ul/minute on a BIAcore CM5 chip containing 260 RU of p75 (channel 2)and 600 RU of trkA (channel 3), and percent binding was determined. Theresults of this analysis are shown in FIG. 11. Increased concentrationsof Fab E3 blocked the interaction of NGF with both p75 and trkA, asshown by decreased signal (measured in RU), indicating that Fab E3blocks the interaction of human NGF with both trkA and p75. Whenantibody E3 (Fab) concentration equaled NGF concentration (at about 2.5nM NGF concentration), no NGF binding was observed (as shown by a signalof zero). The fact that zero percent NGF-receptor binding occurred whenconcentration of NGF was equal to antibody 3E concentration suggestedthat 2.5 nM NGF was at least ten-fold higher than the kD of E3 for NGFand at equilibrium.

Example 2 Evaluation of NGF-Blocking Ability of Anti-NGF AntibodiesUsing Mouse E13.5 Trigeminal Neuron Survival Assay

[0436] The ability of Fab E3 or full antibody E3 to block NGF activitywas evaluated by measurement of the capacity of the antibody to inhibitNGF-dependent survival of mouse E13.5 trigeminal neurons in vitro. Thetrigeminal ganglion is comprised of cutaneous sensory neurons thatinnervate the facial region. The survival of mouse E13.5 trigeminalneurons is a sensitive assay to evaluate the NGF-blocking activity ofanti-NGF antagonist antibodies because NGF is required to supportsurvival of these neurons. For example, at saturating concentrations ofNGF, the survival is close to 100% by 48 hours in culture. By contrast,less than 5% of the neurons survive by 48 hours in absence of NGF.

[0437] The survival assay was conducted as follows: time-mated pregnantSwiss Webster female mice were euthanised by CO₂ inhalation. The uterinehorns were removed and the embryos at embryonic stage E13.5 wereextracted and decapitated. The trigeminal ganglia were dissected usingelectrolytically sharpened tungsten needles. The ganglia were thentrypsinized, mechanically dissociated and plated at a density of 200-300cells per well in defined, serum-free medium in 96-well plates coatedwith poly-L-ornithine and laminin.

[0438] The blocking activity of anti-NGF Fabs or antibodies was assessedby adding to the trigeminal neurons varying doses of anti-NGF antibodiesMab 911 (Fab), 8L2-6D5; H19-L129; E3 and 3C; and human or rat NGF at thefollowing concentrations: 0.4 ng/ml (˜15 pM; this concentrationrepresented a saturating concentration of NGF for survival) and 0.04ng/ml (˜1.5 pM; this concentration is around the IC50). After 48 hoursin culture, the cells were subjected to an automated immunocytochemistryprotocol performed on a Biomek FX liquid handling workstation (BeckmanCoulter) as follows: fixation using 4% formaldehyde, 5% sucrose, andPBS; permeabilization using 0.3% Triton X-100 in PBS); blocking ofunspecific binding sites using 5% normal goat serum, 0.11% BSA in PBS;and sequential incubation with a primary and secondary antibodies todetect neurons. The primary antibody was rabbit polyclonal antibodyagainst the protein gene product 89.5 (PGP9.5, Chemicon), an establishedneuronal phenotypic marker. The secondary antibody was Alexa Fluor 488goat anti-rabbit (Molecular Probes), together with the nuclear dyeHoechst 33342 (Molecular Probes) to label the nuclei of all the cellspresent in the culture. Image acquisition and image analysis wereperformed on a Discovery-I/GenII Imager (Universal Imaging Corporation).Images were automatically acquired at two wavelengths for Alexa Fluor488 and Hoechst 33342, with the nuclear staining being used as referencepoint for the image-based auto-focus system of the Imager, since nuclearstaining is present in all of the wells. Appropriate objectives andnumber of sites imaged per well were selected to cover the entiresurface of each well. Automated image analysis was set up to count thenumber of neurons present in each well after 48 hours in culture basedon their specific staining with the anti-PGP9.5 antibody. Carefulthresholding of the image and application of morphology and fluorescenceintensity based selectivity filter resulted in an accurate count ofneurons per well.

[0439] The results of this experiment demonstrated that Fab E3 blockedNGF activity with a high affinity. The results are shown in FIGS. 4-6,and Table 9.

[0440]FIG. 4 is a graph showing NGF-dependent survival of E13.5 neuronsin the presence of varying concentration of human and rat NGF.

[0441]FIG. 5 is a graph comparing the NGF blocking effect of variousFabs in the presence of either 0.04 ng/ml of human NGF (approximately1.5 pM; shown in the lower panel) or 0.4 ng/ml human NGF (approximately15 pM; shown in the upper panel). 1.5 pM of NGF was around the EC50 ofNGF promoting survival, while 15 pM represented a saturatingconcentration of NGF. Survival of E13.5 mouse trigeminal neurons invarious concentrations of Fab E3; murine 911 Fab; and Fab H19-L129 andFab 8L2-6D5 was assessed as described above. The IC50 (in pM) wascalculated for each Fab at each NGF concentration, and is shown in Table9. Fab E3 strongly blocked human NGF-dependent trigeminal neuronsurvival, with an IC50 of approximately 21 pM in the presence of 15 pMhuman NGF, and an IC50 of approximately 1.2 pM in the presence of 1.5 pMhuman NGF. Fabs 3C and H19-L129 also strongly blocked humanNGF-dependent trigeminal neuron survival.

[0442]FIG. 6 is a graph comparing the NGF blocking effect of variousFabs in the presence of either 0.04 ng/ml of rat NGF (approximately 1.5pM; shown in the lower panel) or 0.4 ng/ml rat NGF (approximately 15 PM;shown in the upper panel). 1.5 pM of NGF was around the EC50, while 15pM represented a saturating concentration of NGF. Survival of E13.5mouse trigeminal neurons in various concentrations of Fab E3; murine Fab911; and Fab H19-L129 and 8L2-6D5 was assessed as described above. TheEC50 (in pM) was calculated for each Fab at each NGF concentration, andis shown in Table 9. Fab E3 strongly blocked human NGF-dependenttrigeminal neuron survival, with an IC50 of approximately 31.6 pM in thepresence of 15 pM rat NGF, and an IC50 of approximately 1.3 pM in thepresence of 1.5 pM rat NGF. Fabs 3C and H19-L129 also strongly blockedrat NGF-dependent trigeminal neuron survival. TABLE 9 IC50 (in thepresence IC50 (in the presence Human NGF of 15 pM NGF) pM of 1.5 pM NGF)pM 8L2-6D5 Fab 1580.5 461.8 H19-L129 Fab 60.1 9.6 3E Fab <21.0 <1.2 3CFab 80.9 5.6 911 Fab 322.3 63.5 IC50 IC50 Rat NGF (15 pM NGF) pM (1.5 pMNGF) pM 8L2-6D5 Fab 730.3 169.4 H19-L129 Fab 31.0 6.0 3E Fab <8.3 <1.33C Fab 31.6 6.0 911 Fab 161.0 34.6

[0443] In a different experiment, we compared the ability of fullantibody E3 and Fab 3E to inhibit NGF-dependent survival of E13.5neurons in the presence of 0.4 ng/ml (saturating concentration) of humanNGF. The results of the analysis are shown in FIG. 12. Full antibody E3and Fab 3E showed similar levels of inhibition of NGF-dependent survivalwhen the concentration of whole antibody and Fab were normalized to thenumber of NGF binding sites (Fab has one binding site and whole antibodyhas two binding sites). These results demonstrated that there was noavidity effect due to the binding of a full antibody to the NGF dimer.

[0444] In another experiments, we compared the ability of variousconcentrations (20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, and 0.0 nM) ofantibody E3, antibody 911, and a trkA receptor immunadhesin (consistingof the extracellular domain of the NGF receptor trkA fused with theimmunoglobulin Fc domain, CH2—CH3) to inhibit NGF-dependent survival ofE13.5 neurons in the presence of 0.4 ng/ml (saturating conditions).These results are shown in FIG. 13. These results demonstrated thatantibody E3 blocked NGF better than either antibody 911 or the trkAimmunoadhesin.

Example 3 Evaluation of the Specificity of Anti-NGF Antibody E3 UsingMouse Trigeminal and Nodose Neuron Survival Assays

[0445] The ability of antibody E3 to specifically block NGF activity wasevaluated by measurement of the capacity of the antibody to inhibitsurvival of mouse E17/18 trigeminal neurons in vitro in the presence ofsaturating concentrations of NGF, the NGF-related neurotrophin NT3, orthe NGF-unrelated neurotrophic factor, macrophage stimulating protein(MSP). The survival of mouse E17/18 trigeminal neurons is a sensitiveassay to evaluate the NGF-blocking activity of anti-NGF antagonistantibodies because NGF is required to support survival of these neuronsat higher concentrations than the level of NGF required to supportsurvival of E13.5 TG neurons). Survival of these neurons is alsosupported by NT3 or MSP; therefore, the survival of these neurons isalso a sensitive assay to evaluate whether the anti-NGF antagonistantibody also blocked NT3 or MSP.

[0446] The ability of antibody E3 to specifically block NGF activity wasalso evaluated by measurement of the capacity of the antibody to inhibitsurvival of mouse nodose E17 neurons in the presence of saturatingconcentrations of BDNF or NT4/5. Survival of nodose neurons is supportedby BDNF or NT4/5; therefore, survival of these neurons is a sensitiveassay to evaluate the BDNF or NT4/5-blocking ability of the anti-NGFantagonist antibody.

[0447] The survival assay was conducted as follows: time mated pregnantSwiss Webster female mice were euthanised by CO₂ inhalation. The uterinehorns were removed and the embryos (at embryonic day 17 or 18) wereextracted and decapitated. The trigeminal and nodose ganglia weredissected and cleaned. The ganglia were then trypsinised, mechanicallydissociated and plated at a density of 100-300 cells per well indefined, serum-free medium in 4-well plates (Greiner) coated withpoly-L-ornithine and laminin.

[0448] E17/18 trigeminal neurons were grown either without addedneurotrophic factors (negative control) or in the presence of saturatingconcentrations of human NGF (400 pM and 15 pM) (positive control); NT3(400 pM); or MSP (600 pM). Duplicate cultures were set up that includedvarying concentrations of E3 and 911 Fabs and full antibodies.Concentration of Fab and full antibodies was indicated per binding site(e.g., a full antibody contains two binding sites, while a Fab containsone binding site).

[0449] E17 nodose neurons were grown either in the absence of addedneurotrophic factors (negative control), or with saturatingconcentrations of BDNF (400 pM) (positive control) or NT4/5 (400 pM) orNGF unrelated growth factor ILF (interleukin inhibitory factor). Highconcentrations of neurotrophins were used, as the goal of thisexperiment was to test specificity of the antibodies. Duplicate cultureswere set up that included varying again with and without the addition ofantibodies E3 and 911. After 48 hours in culture the total number ofneurons surviving in each well under each condition was ascertained bymanual counting using a phase-contrast microscope.

[0450] The results of these experiments demonstrated that E3 and 911antibodies completely blocked the survival promoting effects of NGF onE18 trigeminal neurons. By contrast, E3 and 911 antibodies had no effecton survival of trigeminal neurons promoted by NT3 or MSP, or survival ofnodose neurons promoted by BDNF or NT4/5 or LIF. These resultsdemonstrated that antibody E3 possessed selective specificity for NGF,as there was no detected interaction between these antibodies and otherNGF related neurotrophins (NT3, NT4/5, BDNF) at concentrations 1000-foldto 10,000-fold higher than effective concentration for NGF blocking.Further, these results demonstrated that the neuronal death seen inNGF-supplemented cultures of NGF-dependent neurons on addition ofantibody or Fab E3 was due to a specific interaction between theseantibodies and NGF and was not due to a generalized toxic effect. Mouseanti-NGF antagonist antibody 911 was also tested, and similar resultswere observed. Note that due to the high concentrations of neurotrophinsused, both antibody E3 and 911 are very close to their titrationconditions and were expected to bind NGF at similar levels because thedifferences in binding affinity of these antibodies to NGF would to beless apparent under these conditions.

[0451] The results of these experiments are shown in FIGS. 14, 15, 16,and 17. The data showed mean percent survival after 48 hours in culture(±standard error of mean, n=3 for each data point) relative to thesurvival seen in the positive control for each experiment (e.g., 100%survival of trigeminal neurons grown in the presence of saturating NGFconcentration, and 100% survival of nodose neurons grown in the presenceof saturating BDNF concentration, respectively). FIGS. 14-15 are graphsshowing that anti-NGF antagonist antibody E3 or Fab E3 did not inhibitthe survival promoted by NT3, and MSP, even at antibody concentrationsas high as 200 nM. By contrast, 20 nM of antibody E3 or Fab 3E and Fab911 totally blocked NGF-elicited survival. Mouse anti-NGF antagonistantibody 911 was also tested, and similar results were observed.Specifically, FIG. 14 is a graph showing comparison of the effect ofvarious concentrations (20 nM, 2 nM, or 0.2 nM) of E3 Fab (termed “3E”in the figure) and mouse antibody 911 Fab on survival of E18 trigeminalneurons in the presence of no added neurotrophin (termed “control”), 400pM NGF (termed “NGF-400 pM), 10 nM NT3 (termed “NT3-10 nM) or 600 pM MSP(termed “MSP-600 pM). FIG. 15 is a graph depicting comparison of theeffect of various concentrations (200 nM and 80 nM) of E3 Fab and fullantibody and mouse antibody 911 full antibody and Fab of survival of E17trigeminal neurons in the presence of no added neurotrophins (termed “nofactor”), 400 pM NGF (termed “NGF-400 pM), 10 nM NT3 (termed “NT3-10 nM)or 600 pM MSP (termed “MSP-600 pM).

[0452]FIG. 16-17 are graphs showing that anti-NGF antagonist antibody E3or Fab E3 did not inhibit survival of E17 nodose neurons promoted byBDNF, NT4/5 or LIF. Mouse anti-NGF antagonist antibody 911 was alsotested, and similar results were observed. Specifically, FIG. 16 is agraph showing comparison of the effect of various concentrations (200 nMor 80 nM) of full antibody E3 (termed “3E in the figure”), Fab E3, fullantibody 911, or Fab 911 on the survival of E17 nodose neurons in thepresence of no added neurotrophins (termed “no factors”), 400 pM BDNF(termed “BDNF-400 pM), 400 pM NT4/5 (termed “NT4/5-400 pM), or 2.5 nMLIF (termed “LIP-2.5 nM). FIG. 17 is a graph showing comparison of theeffect of various concentrations (200 nM, 20 nM, 2 nM) of Fab E3 (termed“3E in the figure”), or Fab 911 on the survival of E17 nodose neurons inthe presence of no added neurotrophins (termed “control”), 400 pM BDNF(termed “BDNF-400 pM), 400 pM NT4/5 (termed “NT4/5-400 pM), or 2.5 nMLIF (termed “LIP-2.5 nM).

Example 5 Preparation of Mammalian Expression Vectors and Expression ofAntibody E3 in Mammalian Cells

[0453] Three mammalian expression vectors were designed and constructedfor use in the expression of antibody E3 in mammalian cells.

[0454] Vector Db.911.3E is an expression vector comprising the heavychain variable region of the E3 antibody and the human IgG2a constantregion, and is suitable for transient or stable expression of the heavychain. Db.911.3E consists of nucleotide sequences corresponding to thefollowing regions: the murine cytomegalovirus promoter region(nucleotides 1-612); a synthetic intron (nucleotides 619-1507); the DHFRcoding region (nucleotides 707-1267); human growth hormone signalpeptide (nucleotides 1525-1602); antibody 3E heavy chain variable region(nucleotides 1603-1965); human heavy chain IgG2a constant regioncontaining the following mutations: A330P331 to S330S331 (amino acidnumbering with reference to the wildtype IgG2a sequence; see Eur. J.Immunol. (1999) 29:2613-2624); SV40 late polyadenylation signal(nucleotides 2974-3217); SV40 enhancer region (nucleotides 3218-3463);phage fl region (nucleotides 3551-4006) and beta lactamase (AmpR) codingregion (nucleotides 4443-5300). Db.911.3E was deposited at the ATCC onJan. 8, 2003, and was assigned ATCC Accession No. PTA-4895.

[0455] Vector Eb.911.3E is an expression vector comprising the lightchain variable region of the E3 antibody and the human kappa chainconstant region, and is suitable for transient expression of the lightchain. Eb.911.3E consists of nucleotide sequences corresponding to thefollowing regions: the murine cytomegalovirus promoter region(nucleotides 1-612); human EF-1 intron (nucleotides 619-1142); humangrowth hormone signal peptide (nucleotides 1173-1150); antibody E3 lightchain variable region (nucleotides ¹²⁵I-1571); human kappa chainconstant region (nucleotides 1572-1892); SV40 late polyadenylationsignal (nucleotides 1910-2153); SV40 enhancer region (nucleotides2154-2399); phage fl region (nucleotides 2487-2942) and beta lactamase(AmpR) coding region (nucleotides 3379-4236). Eb.911.3E was deposited atthe ATCC on Jan. 8, 2003, and was assigned ATCC Accession No. PTA-4893.

[0456] Vector Eb.pur.911.3E is an expression vector comprising the lightchain variable region of the E3 antibody and the human kappa constantregion, and is suitable for stable expression of the light chain.Eb.pur.911.3E consists of nucleotide sequences corresponding to thefollowing regions: the murine cytomegalovirus promoter region(nucleotides 1-612); human EF-1 intron (nucleotides 619-1758); pac gene(puromycinR) coding region (nucleotides 739-1235); human hsp70 5′UTRregion (nucleotides 1771-1973); human growth hormone signal peptide(nucleotides 1985-2062); antibody E3 light chain variable region(nucleotides 2063-2383); human kappa chain constant region (nucleotides2384-2704); SV40 late polyadenylation signal (nucleotides 2722-2965);SV40 enhancer region (nucleotides 2966-3211); phage f1 region(nucleotides 3299-3654) and beta lactamase (AmpR) coding region(nucleotides 4191-5048). Eb.pur.911.E3 was deposited at the ATCC on Jan.8, 2003, and was assigned ATCC Accession No. PTA-4894.

[0457] Transient cell expression was perfomed as follows: CHO andHEK293T cells in 150 mm dishes were transiently co-transfected with 25ug of each plasmid (i.e., one plasmid containing the heavy chain and oneplasmid containing the light chain). DNA was mixed with 100 ullipofectamine 2000 (Invitrogen) according to the manufacturer'sinstructions. The DNA-lipid complexes were allowed to contact the cellsin DMEM/F12 medium without serum or antibiotics for 5 hours. Followingthis incubation, the media was changed for expression to Opti-MEM(Invitrogen) without any additives for two days. Cell supernatantscontaining antibody were harvested sequentially up to four times withsubsequent media replacement. Supernatants were purified by affinitychromatography using MapSelect Protein A resin (Amersham biosciences17-5199-O₂). Antibody was bound to the protein A resin in 0.3M glycine,0.6M NaCl buffer at pH 8, then eluted with 0.1 M citrate buffer at pH 3.Fractions containing antibody were immediately neutralized with 1M Trisbyffer at pH 8.0, Antibody fractions were then dialyzed and concentratedin PBS.

Example 6 Anti-NGF Antibody E3 is Effective in Treating Post-SurgicalPain

[0458] We used a pain model that mimics post surgical pain to assess theefficacy of treatment with antibody E3. Antibody E3 comprised the humanheavy chain IgG2a constant region containing the following mutations:A330P331 to S330S331 (amino acid numbering with reference to thewildtype IgG2a sequence; see Eur. J. Immunol. (1999) 29:2613-2624); thehuman light chain kappa constant region; and the heavy and light chainvariable regions shown in Tables 1A and 1B.

[0459] Animals. Male Sprague Dawley rats weighting between 220-240 gramswere purchased from Harlan (Wisconsin) and acclimated to the animalfacility for one week prior to surgery.

[0460] Surgery. The surgery was based on the procedure described byBrennan, et al. Pain 64:493-501 (1996). Animals were anesthetized with a2% isoflurane in air mixture that was maintained during surgery via anose cone. The plantar surface of the right hind paw was prepared with apovidone-iodine pad, and a 1-cm central longitudinal incision was madethrough skin and fascia, starting 0.5 cm from the edge of the heel andextending toward the toes. Measurements were made with a ruler with thefoot held in a flexed position. The plantaris muscle was elevated usingcurved forceps and incised longitudinally. The muscle was incisedthrough its full depth, between the origin and insertion. Bleeding wascontrolled throughout surgery by pressure applied through a gauze pad.The wound was closed with two mattress sutures (5-0 ethilon blackmonofilament). These sutures were knotted 5-6 times, with the first knotloosely tied. The wound site was swabbed with bacitracin solution.Animals were allowed to recover and rest in clean cages for two hours ormore before behavioral testing began.

[0461] Evaluating resting pain. A cumulative pain score was used toassess pain related to weight bearing. Animals were placed on a plasticmesh (grid: 8 mm²) in clear plastic cages that were elevated on aplatform (h: 18″) allowing inspection of the underside of their paws.After a 20 minute acclimation period, weight bearing was assessed on ascale of 0 to 2. A score of 0 was given if the paw was blanched orpressed against the mesh, indicating full weight bearing. A score of 1was given if the paw was favored with the skin just touching the mesh,with no blanching or indentation of the skin. A score of 2 was given ifthe paw was held completely off the mesh. Flinching the paw wasconsidered a 2 if the rat was still at rest. Each animal was observedfor 1 minute every 5 minutes for 30 minutes. The sum of 6 scores (0-12)obtained during ½-hour was used to assess pain in the incised foot.Frequency of scores of 2 was also calculated and used to assess theincidence of severe pain or total guarding of the paw by the animal.Each animal was tested 24 hours before surgery (baseline), and 2 h, 24h, 48 h, and 72 h postoperatively. The results of this experiment areshown in FIG. 1, which depicts the cumulative resting pain scoreobserved in animals treated with 35 mg/kg of anti-NGF mouse antibody911. These results demonstrated that treatment with anti-NGF antibodysignificantly reduced post-surgical resting pain. Weight bearing was agood correlate of how willing the animal was to use the limb, andtherefore was an effective measure of pain relief.

[0462] The E3 antibody was injected intra peritoneal (i.p.) at variousconcentrations of the antibody (0.004, 0.01, 0.02, 0.1, 0.6, and 1 mgper kilogram of animal weight) at 15 hours pre-incision. The negativecontrol group received no antibody but was injected i.p. with a salinesolution. Fentanyl at 0.01 mg/kg was injected i.p. as a positive control30 minutes before testing at 24 hours post-surgery. Each experimentinvolved 8 animals (n=8 per group) for each condition, and the controlgroup had 56 animals. Surgery was performed and a cumulative pain scorewas measured as described above. Resting pain was evaluated twenty-fourhours after the surgery.

[0463] As shown in FIG. 7, humanized anti-NGF antibody E3 significantlyreduced resting pain (p<0.05) after surgery when administered at 0.02mg/kg to 1 mg/kg dosage. A “*” denotes a significantly significantdifference from control (p<0.05). Treatment with 0.02 mg/kg alleviatedpain behavior at least as effectively as treatment with 0.01 mg/kgfentanyl. This dose of fentanyl is 10 times the normal human dose ofthis potent opioid.

[0464] In another experiment, the efficacy of the E3 antibody inreducing post-surgical pain when administered post-surgically wastested. Antibody E3 (0.5 mg/kg) were injected intravenously (i.v.) twohours after surgery. The control group received no antibody but wasinjected i.v. with a saline solution. Surgery was performed and restingpain expressed as a cumulative pain score was assessed 24 hours aftersurgery. As shown in FIG. 8, treatment with anti-NGF antibodysignificantly (p<0.05) reduced resting pain at twenty-four hours afterincision when the antibody was administered 2 hours post-incision. Theseresults demonstrated that E3 antibody effectively alleviatedpost-surgical pain when administered after surgery.

Example 7 Assessment of Analgesic Effects of Anti-NGF AntagonistAntibody 911 in a Rat Model of Rheumatoid Arthritis

[0465] The analgesic effects of anti-NGF antibody, 911 (see Hongo etal., Hybridoma 19(3):215-227 (2000)) in complete Freund's adjuvant(CFA)-induced chronic arthritis in rats were investigated using thevocalization test, in comparison with indomethacine used as referencesubstance.

[0466] Fifty (50) male Lewis rats (LEWIS LEW/Crl Ico) (Charles RiverBelgium) weighing 150 g to 220 g at the beginning of the experimentalphase were included in this study. All animals were kept for at least 5days before the experiment, and were housed in a temperature (19.5-24.5°C.), relative humidity (45-65%) and 12-h light/dark cycle-controlledroom with ad libitum access to filtered tap-water and standard pelletedlaboratory chow (U.A.R., France) throughout the study. Animals wereindividually identified on the tail.

[0467] On day 0 (DO), arthritis was induced in rats by intradermalinjection into the tail of 0.05 ml of a Mycobacterium butyricum (Difco,USA) suspension in mineral oil (10 mg/ml). On day 14 (D14), arthriticrats were included in the study according to their ability to vocalizeupon gentle flexion of the hindpaw and by their arthritis index,evaluated using an inflammation score for each hind and forepaw (seeKuzuna et al., Chem. Pharm. Bull. (Tokyo) 23:1184-1191 (1975); Pearsonet al., Arthritis Rheum. 2:440-459 (1959)). Animals were scored based onthe following criteria: Score 0: normal aspect; Score 1: erythema; Score2: erythema with slight edema; Score 3: strong inflammation withoutankylosis; Score 4: ankylosis. Only animals able to vocalize upon gentleflexion and presenting a score of 2 or 3 were included in the study.

[0468] Four groups of 10 rats each were included in the study. For group1 (vehicle), on day 14 (D14), after selection, rats were intravenouslyadministered by vehicle (saline). On day 18 (D18), the nociceptiveintensity was evaluated by gentle flexion of the hindpaw and theintensity of the level of vocalization was recorded for each animal. Forgroup 2 (4 days), on D14, after selection, rats were intravenouslyadministered 911 (10 mg/kg). On day 18 (D18), the nociceptive intensitywas evaluated by gentle flexion of the hindpaw and the intensity of thelevel of vocalization was recorded for each animal. For group 3 (24hours), on day 17 after injection of CFA, rats were intravenouslyadministered 911 (10 mg/kg). The nociceptive intensity was evaluated bygentle flexion of the hindpaw 24 hours later, and the intensity of thelevel of vocalization was recorded for each animal. For group 4(indomethacin), on day 18 (D18), the nociceptive intensity was evaluatedby gentle flexion of the hindpaw one hour after oral administration ofindomethacin (10 mg/kg). The intensity of the level of vocalization wasalso recorded for each animal. The test substances were administered ina blind and random manner by intravenous route under a volume of 5ml/kg, whereas indomethacin was administered by oral route under avolume of 10 ml/kg.

[0469] The analgesic effects of anti-NGF antibody 911 are shown in Table10. The results were expressed for each group as the nociceptiveintensity evaluated the intensity of the level of vocalization recordedfor each animal in mV (mean±SEM), and the percentage of variation of thenociceptive intensity calculated from the mean value of thevehicle-treated group. Statistical significance between the treatedgroups and the vehicle group was determined with a Dunnett's test usingthe residual variance after a one-way analysis of variance (P<0.05).TABLE 10 Analgesic effects of 911 in complete freund's adjuvant-inducedchronic arthritis in rats Substances Indomethacin (Day of dosing)Vehicle (D14) 911 (D14) 911 (D17) (D18) Dose (mg/kg) 10 10 10Nociceptive 971.0 ± 116.2 234.7 ± 34.4 * 247.2 ± 41.8 * 145.8 ± 29.9 *intensity (mV) % variation — −76 −75 −85

[0470] As shown in Table 10, anti-NGF antibody 911 significantly reducedpain in a rat model of rheumatoid arthritis 24 hours or 4 days after asingle administration of the antibody.

Example 8 Pharmacological Effects of Anti-NGF Antagonist Antibody E3 and911 in a Rat Model of Rheumatoid Arthritis

[0471] Pharmacological effects (anti-inflammatory and analgesic effects)of anti-NGF antagonist antibody E3 and 911 were investigated in a modelof complete Freund's adjuvant (CFA)-induced chronic arthritis in rats incomparison with indomethacin used as an internal positive controlsubstance. Analgesic effects of E3 and 911 were evaluated by themeasurement of nociceptive response. Anti-inflammatory effects wereevaluated by paw volume, arthritis index (inflammation score), body andhindpaws weight. Paw cytokine levels (IL-6, IL-10, TNF-α and TGF-β11),circulating TGF-β1 in serum, E3 and 911 plasma concentrations,biological parameters and X-ray radiographies were performed at the endof experiment.

[0472] Experimental Protocol

[0473] 1. Study Design

[0474] 80 male Lewis rats (LEWIS Lew/Ico) (Charles RiverLaboratories—Belgium) 5-weeks old were included in this study. They werehoused in a temperature (19.5-24.5° C.) and relative humidity (45-65%)controlled room with a 12-h light/dark cycle, with ad libitum access tofiltered tap-water and standard pelleted laboratory chow (SAFE, France)throughout the study. Upon receipt at animal facilities, they werehoused 5 per cage and a 10-day acclimatization period were observedbefore any testing. Animals were individually identified on the tail.

[0475] Five groups of 10 animals (5-weeks old male Lewis rats—LEWISLew/Ico, from Charles River Laboratories—Belgium) each were included inthis study: Group 1: non arthritic rats/saline (vehicle), i.v. bolus,n=10; Group 2: arthritic rats/saline (vehicle), i.v. bolus, n=10; Group3: arthritic rats/Indomethacin 3 mg/kg, p.o daily over 10 days, n=10;Group 4: arthritic rats/E3, 1 mg/kg, i.v. bolus, n=10; Group 5:arthritic rats/911, 10 mg/kg, i.v. bolus, n=10. The doses were expressedin terms of free active substance (mg/kg). E3 and 911 wereextemporaneously prepared in saline from the stock solution to thedesired concentration. E31 mg/kg: 3.41 mL of the stock solution (0.88mg/ml) q.s.p. 15 mL of saline. 91110 mg/kg: 12 mL of the stock solution(2.5 mg/ml) q.s.p. 15 mL of saline. All diluted solutions (before i.v.injection) were sterilized using a sterile filter unit of 0.20 μm. pHand osmolarity values of diluted solutions were measured before eachi.v. injection. Before the first i.v., osmolarity (mosm/L) for saline,E3, and 911 were 278, 269, and 308 respectively; pH for saline, E3, and911 were 5.93, 6.76, 6.71 respectively. Before the second i.v.,osmolarity (mosm/L) for saline, E3, and 911 were 280, 270, and 309respectively; pH for saline, E3, and 911 were 5.86, 6.72, and 6.59respectively.

[0476] E3 or 911 or saline were administered by i.v. bolus injection onDay 14 and Day 19 after arthritis induction in a coded and random orderwith a volume of 5 mL/kg. The non arthritic group was given by i.v.bolus injection of saline on Day 14 and Day 19 with a volume of 5 mL/kg.Indomethacin was extemporaneously prepared in 1% methylcellulose.Indomethacin was administered by oral route (p.o.) once daily over 10days from Day 14 to Day 23 after arthritis induction in a coded andrandom order with a volume of 10 mL/kg.

[0477] 2. Induction of Arthritis

[0478] On Day 0 (D 0), arthritis was induced in 70 rats by intradermalinjection into the tail of 0.05 ml of a Mycobacterium butyricumsuspension. A group of 10 rats did not receive any intradermal injection(non arthritic rats). On Day 14 (D 14), the arthritic rats were includedin the study using the following criteria: all included rats displayedan increase of mean paw volume (mean of the left and right paw volume)of at least 0.30 ml compared to the mean paw volume (mean of the leftand right paw volume) in the non arthritic group (paw volume measurementas described below); all included rats displayed a vocalization upongentle flexion (nociceptive response measurement as described below);and all included rats displayed a score of arthritis index of 2-3 oneach hindpaw (arthritis index measurement as described below) (theanimals with a score of 0, 1 or 4 were discarded).

[0479] 3. Body Weight

[0480] The animals were weighed once daily from Day 0 to Day 24 (exceptduring the week-end days before the treatment: D 1, D 2, D 8, D 9, D10).All measurements were performed between 9:00 and 12:00 am except at D 14(7:30-9:00 am) and D 24 (7:30-8:00 am).

[0481] 3. Paw Volume Measurement

[0482] The right and left hindpaw volume of each rat (arthritic and nonarthritic rats) was measured using a plethysmometer. The measurementswere performed at the following times (after induction of arthritis):Day 14 (before i.v. bolus or p.o. administration); and Day 24 (5 daysafter the last i.v. bolus injection or 24 h after the last p.o.administration). All measurements were performed between 9:00 and 12:00am. All the data were collected and stored by the WinDas software.

[0483] 4. Arthritis Index

[0484] Arthritis index was evaluated using an inflammation score foreach hind and forepaw (arthritic rats): Score 0: normal aspect; Score 1:erythema; Score 2: erythema with slight edema; Score 3: stronginflammation without ankylosis; Score 4: ankylosis. This evaluation wasperformed at the following times (after induction of arthritis): Day 14(before i.v. bolus or p.o. administration); and Day 24 (5 days after thelast i.v. bolus injection or 24 h after the last p.o. administration).All measurements were performed between 2:00 and 3:00 pm (D 14), 8:00and 9:00 am (D 24). All the data were collected and stored by the WinDassoftware.

[0485] 5. Measurement of Nociceptive Response (Vocalization Test)

[0486] The nociceptive response was evaluated by gentle flexion of theright and left hindpaw repeatedly 2 times at intervals of 4 to 5 secwith a finger of the operator (arthritic rats). The intensity of thelevel of vocalization was recorded for each animal for each hindpaw (2times: on right hindpaw: s1 and s3; 2 times: on left hindpaw: s2 ands4). This evaluation was performed at the following times (afterinduction of arthritis): Day 14 (before i.v. bolus or p.o.administration); Day 18 (before the second i.v. bolus injection or 1 hrafter p.o. administration); and Day 24 (5 days after the last i.v. bolusinjection or 24 h after the last p.o. administration). All measurementswere performed between 9:00 and 12:00 am except at D 14 (7:30-9:00 am)and D 24 (7:30-9:00 am).

[0487] 6. Blood Collection for Measurement of E3 or 911 Concentrationand Circulating TGF-β1 and Hematological Parameters

[0488] On Day 24 (after paw volume and arthritis index measurements andtest vocalization), under general anaesthesia using isoflurane (in amixture of oxygen and nitrous oxide), the blood samples (about 800-1000μl) was collected by capillary action with a micropipette fromretroorbital sinus.

[0489] Measurement of E3 or 911 concentration (groups 2, 4 and 5): Apart of blood sample was collected in tubes containing Li-Heparin(maintained on ice) and centrifuged at 2500-3000 g for 10 min. Plasmasamples (at least 100 μL) were obtained, frozen in liquid nitrogen,stored at −80° C. One sample was slightly hemolyzed (vehicle-treatedarthritic rat # 36).

[0490] Measurement of circulating TGF-β1 (groups 1-2-3-4-5): A part ofblood sample was collected in micro tubes for serum preparation atambient temperature. Following sample collection, blood was mixed andallowed to clot for 30 minutes prior to the centrifugation. The tubeswere centrifuged at about 6000 g for 3 minutes. Each serum sample (atleast 100 μL, except for rat #52 and #53) was aliquoted and stored at−20° C. until sample activation for TGF-β analysis. These aliquots (50vials) were kept for a period of 6 months starting from the end of thestudy. Some samples were slightly hemolyzed (vehicle-treated nonarthritic rat: #2, #5, #9, #10; vehicle treated arthritic rat: #53, #63;E3-treated arthritic rat #31, #51; 911-treated arthritic rat: #52, 62,#64). TGF-β1 levels were measured using human TGF-β1 ELISA kit (ref.DB100, Batch 212258 and 213610, R&D Systems—France).

[0491] Blood collection for hematological parameters (groups1-2-3-4-5:50 vials): A part of blood sample was collected in tubescontaining K3-EDTA (at least 100 μL). The determination of parameterswere performed on the day of the collection and the samples were notstored. The hematological parameters including red blood cells, whiteblood cells, platelets, hemoglobin, hematocrit were measured with ahematology cell counter (D 24). Some hematological parameters were notmeasured due to the clotted samples (vehicle-treated non arthritic rat:#10; E3-treated arthritic rats: #59, #67; 911-treated arthritic rats:#16).

[0492] 7. Paw Cytokines Levels

[0493] On Day 24 (5 days after the last i.v. bolus injection or 24 hoursafter the last p.o. administration) (after X-rays radiographies), eachanimal hindpaw (arthritic and non arthritic rats) was weighed and wascollected in a labelled polyethylene vial. Tissue samples were frozen inliquid nitrogen and stored at −80° C.

[0494] Preparation of joint homogenates: Frozen hind paws werepulverized using a Bio-Pulverizer. The powdered hind paws were thenplaced into a 50 ml conical centrifuge tube containing 3 ml PBSsupplemented with 50 μl of anti-protease cocktail and homogenized on iceusing Ultra-Turrax homogenizer (50% of the maximal speed). Homogenateswere then centrifuged at 2000×g for 15 minutes at 4° C. and supernatantswere filtered through 0.2 μm Sartorius filters, aliquoted and stored at−80° C. until use.

[0495] Cytokine levels measurement: Cytokine levels of TNF-α (Rat TNF-αELISA kit, ref. RTA00, Batch 213718, R&D Systems, France), IL-1βRatIL-1β (ELISA kit, ref. RLB00, Batch 212435, R&D Systems, France), IL-6Rat IL-6 ELISA kit, ref. R6000, Batch 211773, 214008 and 214362, R&DSystems, France), and TGF-βHuman TGF-β1 ELISA kit, ref. DB100, Batch212258 and 213610, R&D Systems, France) were determined in duplicate,according to the manufacturer's procedure. Aliquots of hind pawhomogenates were stored at −80° C.

[0496] 8. X-Ray Analysis

[0497] On Day 24, after blood collecting the animals were sacrificed andX-ray radiographies (hindpaws) were obtained for assessment of jointlesions. X-ray analysis was focused on articular erosions, articularspace, periosteum abnormalities on both hindpaws. All the radiographieswere analyzed by looking at seven different items: the soft tissuedamage, deformity, demineralization, joint space, erosions, osteogenesisand periostal reaction. For each animal, the first six items wereanalyzed independently by looking at the worse hind foot. The periostalreaction was analyzed by looking at the tail. For each item, the scoregoes from 0 (normal) to 4 (maximal damage). Therefore the total scoregoes from 0 to 28. The radiographic interpretation was done by the samereader without knowing anything about the animals (treated or nottreated).

[0498] 9. Observations

[0499] One animal (#65) died at D 23 after indomethacin administration(before the administration at D 23) due to an unknown cause.

[0500] 10. Analysis and expression of results

[0501] All results were reported as Mean±S.E.M. of 10 rats in each groupat each time point. Paw volume was expressed in ml calculated from themean value of the right and left paw volume. Arthritis index wascalculated from the sum of the score obtained for each of the 4 paws.The nociceptive response was evaluated by the intensity of the level ofvocalization recorded for each animal (mean of 4 values: 2 times/paw) inmV. The percentage inhibition of the nociceptive response was calculatedfrom the mean value of the vehicle-treated arthritic group [(mean valueof vehicle-treated arthritic group-mean value of treated arthriticgroup/mean value of vehicle-treated arthritic group)*100]. Body weightwas expressed in grams. Hindpaws (left and right) weight was expressedin grams. Cytokine levels (IL-6, IL-1β, TNF-α and TGF-β1) of each hindpaw was expressed in pg/ml. Circulating levels of TGF-β1 was expressedin pg/ml. Radiological index for each parameter (demineralization,erosions, periostal reaction, soft tissue damage, space joint,osteogenesis deformity) and total radiological index (total score) werecalculated from the sum of the scores obtained for each parameter. Theinter-group significances of the deviations between the values ofvehicle-treated group (arthritic rats) and vehicle-treated group (nonarthritic rats) were assessed by the Student t test or Mann-Whitney RankSum Test when equal variance or normality test failed. The inter-groupsignificances of the deviations between the values of vehicle-treatedgroup (arthritic rats) and E3- and 911- and Indomethacin-treated groupswere assessed by the 1-way analysis of variance ANOVA followed by thenon-paired Dunnett t test. A probability of P≦0.05 was considered assignificant. All statistical analysis was performed by the Sigmastat™software.

[0502] Results

[0503] 1. Nociceptive Response (Vocalization Test)

[0504] As shown in Table 11 and FIG. 18, on D 14, the nociceptiveresponse was 4147±331, 4386±235, 4644±367 and 4468±143 in vehicle-,indomethacin-, E3-, and 911-treated arthritic groups, respectively.Indomethacin strongly and significantly decreased the nociceptiveresponse after 3 mg/kg/day p.o. (for 10 days) by about −3768 mV (%inhibition: 71%) and −4353 mV (% inhibition: 74%) at D 18 and D 24,respectively compared to the vehicle-treated arthritic group (D 18:1511±398 vs 5279±326 mV; D 24: 1552±508 vs 5905±345 mV). E3 (1 mg/kgi.v. at D 14 and D 19) strongly and significantly decreased thenociceptive response by about −4167 mV (% inhibition: 79%) and −5905 mV(% inhibition: 100%) at D 18 and D 24, respectively compared to thevehicle-treated arthritic group (D 18: 1112±401 vs 5279±326 mV; D 24:0±0 vs 5905±345 mV). 911 (10 mg/kg i.v. 2 days at D 14 and D 19)strongly and significantly decreased the nociceptive response by about−3932 (% inhibition: 74%) and −5358 mV (% inhibition: 91%) at D 18 and D24, respectively compared to the vehicle-treated arthritic group (D 18:1347±492 vs 5279±326 mV; D 24: 547±307 vs 5905±345 mV). TABLE 11 Effectsof E3 and 911 after i.v. injection (2 days: D 14-D 19) on nociceptiveresponse in rheumatoid arthritis in rats Day D14 D 18 D 24 Arthriticvehicle i.v. 4147 ± 331 5279 ± 326 5905 ± 345 Rats E3 1 mg/kg i.v. 4644± 367 1112 ± 401 *   0 ± 0 * % inhibition   0  79  100 911 10 mg/kg i.v.4468 ± 143 1347 ± 492 *  547 ± 307 * % inhibition   0  74  91Indomethacin 4386 ± 235 1511 ± 398 * 1552 ± 508 3 mg/kg p.o. (over 10days) % inhibition   0  71  74

[0505] 2. Body Weight

[0506] As shown in Table 12 and FIG. 19, a marked decrease in the bodyweight gain was observed in arthritic rats in comparison to nonarthritic rats from D 0 to D 14 due to arthritis establishment. At D 14(selection day) the arthritic rats displayed a significant decrease inweight compared to the non arthritic rats (289±2 vs 217±4 g) (Student ttest P<0.05). However, no significant difference in weight (D 14) wasdetected in all arthritic groups (Dunnett t test P>0.05). The bodyweight moderately and significantly increased in Indomethacin-treatedgroup (3 mg/kg/day for 10 days) from D 17 to D 24 with a maximum ofabout 43 g at D 24 compared to the vehicle-treated arthritic group(261±5 vs 218±3 g). After E3 treatment (1 mg/kg i.v. at D 14 and D 19),the body weight moderately and significantly increased from D 17 to D 24with a maximum of about 46 g at D 24 compared to the vehicle-treatedarthritic group (264±5 g vs 218±3 g). After 911 treatment (10 mg/kg i.v.at D 14 and D 19), the body weight moderately and significantlyincreased from D 18 to D 24 with a maximum of about 47 g at D 24compared to the vehicle-treated arthritic (265±7 vs 218±3 g). TABLE 12Effects of E3 and 911 after i.v. injection (2 days: D 14-D 19) on bodyweight in rheumatoid arthritis in rats Day D0 D3 D4 D5 D6 D7 D11 D12 D13D14 Non vehicle i.v. 197 ± 2 215 ± 2 222 ± 2 232 ± 2 236 ± 2 244 ± 2 272± 2 277 ± 2 282 ± 2 289 ± 2 Arthritic Rats Arthritic vehicle i.v. 199 ±2 214 ± 2 221 ± 2 230 ± 2 236 ± 2 241 ± 3 229 ± 6 223 ± 5 218 ± 5 217 ±4 Rats E3 1 mg/kg i.v. 206 ± 4 222 ± 3 230 ± 3 241 ± 3 243 ± 3 249 ± 3242 ± 6 237 ± 6 230 ± 5 225 ± 5 911 10 mg/kg i.v. 201 ± 2 211 ± 5 218 ±5 227 ± 5 231 ± 5 239 ± 5 234 ± 8 228 ± 7 221 ± 7 218 ± 6 Indomethacin202 ± 3 217 ± 4 225 ± 4 235 ± 4 239 ± 4 246 ± 4 242 ± 7 235 ± 7 227 ± 6224 ± 5 3 mg/kg p.o. over 10 days Day D15 D16 D17 D18 D19 D20 D21 D22D23 D24 Non vehicle i.v. 285 ± 2 291 ± 2 297 ± 2 302 ± 3 307 ± 3 308 ± 3312 ± 3 316 ± 3 321 ± 3 326 ± 3 Athritic Rats Arthritic vehicle i.v. 213± 4 212 ± 4 211 ± 3 210 ± 3 208 ± 3 210 ± 3 212 ± 3 214 ± 3 216 ± 3 218± 3 Rats E3 1 mg/kg i.v. 223 ± 5 224 ± 5 227 ± 4 * 232 ± 4 * 235 ± 4 *238 ± 4 * 245 ± 4 * 250 ± 5 * 257 ± 5 * 264 ± 5 * 911 10 mg/kg i.v. 217± 5 221 ± 5 226 ± 5 229 ± 5 * 233 ± 6 * 239 ± 6 * 246±6 * 253 ± 6 * 258± 6 * 265 ± 7 * Indomethacin 230 ± 4 230 ± 5 231 ± 4 * 234 ± 4 * 236 ±4 * 241 ± 4 * 246 ± 4 * 248 ± 5 * 253 ± 5 * 261 ± 5 * 3 mg/kg p.o. over10 days

[0507] Values are expressed in grams as Mean±S.E.M. n=10 animals pergroup except at D 23 and D 24 (n=9) for Indomethacin

[0508] Dunnett t test: * P≦0.05 vs vehicle-treated arthritic rats

[0509] 3. Paw Volume

[0510] On D 14, a randomization was performed in order to obtainhomogenous groups in terms of paw volume. As shown in Table 13, on D 14,the hindpaw volume (mean of the right and left paw volume) wassignificantly greater in arthritic group than that in non arthriticgroup (2.10±0.05 vs 1.44±0.02 mL (Student t test P<0.05)). Indomethacin(3 mg/kg/day p.o. for 10 days) significantly decreased the paw volume byabout −0.75 mL (D 24) compared to the vehicle-treated arthritic group(1.59±0.03 mL vs 2.34±0.08 mL). E3 (1 mg/kg i.v. on D 14 and D 19)slightly and significantly increased the paw volume by about 0.37 mLcompared to the vehicle-treated arthritic group (2.71±0.09 mL vs2.34±0.08 mL). 911 (10 mg/kg i.v. on D 14 and D 19) slightly andsignificantly increased the paw volume by about 0.36 mL compared to thevehicle-treated arthritic group (2.70±0.11 mL vs 2.34±0.08 mL). TABLE 13Effects of E3 and 911 after i.v. injection (2 days: D 14-D 19) on pawvolume in rheumatoid arthritis in rats Day D14 D24 Non vehicle i.v. 1.44± 0.02 1.47 ± 0.02 Arthritic Rats Arthritic vehicle i.v. 2.10 ± 0.052.34 ± 0.08 Rats E3 1 mg/kg i.v. 2.06 ± 0.03 2.71 ± 0.09 * 911 10 mg/kgi.v. 2.02 ± 0.07 2.70 ± 0.11 * Indomethacin 2.08 ± 0.06 1.59 ± 0.03 * 3mg/kg p.o. over 10 days

[0511] 4. Arthritis Index

[0512] As shown in Table 14, on D 14, the arthritis index was 10.1±0.8,8.7±0.6, 10.2±0.4 and 9.4±0.7 and in vehicle- indomethacin-, E3-, and911-treated arthritic groups, respectively. Indomethacin strongly andsignificantly decreased the arthritis index after 3 mg/kg/day p.o. (for10 days) by a maximum of about −8.0 compared to the vehicle-treatedarthritic group (2.7±0.7 vs 10.7±0.6). E3 (1 mg/kg i.v. on D 14 and D19) did not affect the arthritis index compared to the vehicle-treatedarthritic group (11.4±0.4 vs 10.7±0.6). 911 (10 mg/kg i.v. on D 14 and D19) did not affect the arthritis index compared to the vehicle-treatedarthritic group (10.9±0.7 vs 10.7±0.6). TABLE 14 Effects of E3 and 911after i.v. injection (2 days: D 14-D 19) on arthritis index inrheumatoid arthritis in rats Day D14 D24 Arthritic vehicle i.v. 10.1 ±0.8 10.7 ± 0.6 Rats E3 1 mg/kg i.v. 10.2 ± 0.4 11.4 ± 0.4 911 10 mg/kgi.v.  9.4 ± 0.7 10.9 ± 0.7 Indomethacin  8.7 ± 0.6  2.7 ± 0.7 * 3 mg/kgp.o. over 1O days

[0513] 5. Paw Cytokines Levels

[0514] As shown in Table 15, on D 24, the left and right paws cytokinelevels were increased in arthritic vehicle-treated group by a maximum ofabout 3.5 (IL-1β), 4 (TNF-α) and 1.8 (TGF-β1) fold compared to thenon-arthritic vehicle-treated group. No significant difference wasobserved for IL-6 levels, in right and left paw, between the two groups.The cytokines levels of arthritic group were similar in left and rightpaw: 259.7±38.5 vs 219.2±32.4, 4802.8±365.5 vs 4007.1±380.4, 17.8±1.6 vs18.6±1.9 and 9735.0±1219.8 vs 9161.4±846.1 pg/ml for IL-6, IL-β, TNF-αand TGF-β1 respectively. Indomethacin slightly, but significantly,decreased the TGF-β1 level in right paw after 3 mg/kg/day p.o. (for 10days) by about 1.3 times, compared to the vehicle-treated arthriticgroup (7057.4±335.6 vs 9161.4±846.1), whereas it did not modify IL-6,TNF-α or IL-1β levels. A similar but not significant effect was observedin the left paw. E3 (1 mg/kg i.v. on D 14 and D 19) did not affect theIL-6, IL-1β, TNF-α or TGF-β1 levels, in both paws, compared to thevehicle-treated arthritic group. 911 (10 mg/kg i.v. on D 14 and D 19)increased the IL-1β level in right paw compared to the vehicle-treatedarthritic group (6215.3±666.7 vs 4007.1±380.4). It had no effect onothers cytokine levels in both paws. TABLE 15 Effect of E3 and 911 afteri.v. injection (2 days on D 14 and D 19) on paw cytokines levels inrheumatoid arthritic rats Arthritic Rats Non-arthritic Rats E3 911Indomethacin vehicle i.v. vehicle i.v. 1 mg/kg i.v. 10 mg/kg i.v. 3mg/kg p.o. Left paw cytokines levels IL-6  298.6 ± 35.6  259.7 ± 38.5 234.4 ± 35.2  262.5 ± 42.5  249.7 ± 60.4 IL-1β 1383.0 ± 57.9 4802.8 ±365.5 5060.0 ± 473.5  5500.8 ± 625.3 4029.1 ± 449.9 TNF-α   4.3 ± 2.9 17.8 ± 1.6  23.6 ± 2.5   29.9 ± 4.8  29.9 ± 3.6 TGF-β1 5264.7 ± 209.29735.0 ± 1219.8 9796.7 ± 491.2 11053.5 ± 713.3 7708.2 ± 293.9 Right pawcytokines levels IL-6  286.4 ± 76.1  219.2 ± 32.4  214.6 ± 47.2  284.9 ±38.9  295.9 ± 47.8 IL-1β 1342.1 ± 86.1 4007.1 ± 380.4 4853.5 ± 605.0 6215.3 ± 666.7 * 3884.4 ± 534.4 TNF-α  15.7 ± 4.8  18.6 ± 1.9  21.5 ±2.5   33.4 ± 5.7  30.6 ± 5.7 TGF-β1 5024.8 ± 148.4 9161.4 ± 846.1 9362.7± 423.4 10861.2 ± 604.6 7057.4 ± 335.6 *

[0515] Values are expressed in pg/ml, as Mean±S.E.M.

[0516] n=10 animals per group except for Non-arthritic/vehicle (Rightpaw), Arthritic/vehicle (Left paw) and Indomethacin (n=9)

[0517] Dunnett t test: * P≦0.05 vs vehicle-treated arthritic rats

[0518] 6. Measurement of Circulating TGF-β1

[0519] As shown in Table 16, on D 24, the serum TGF-β1 level wasincreased in arthritic vehicle-treated group compared to the nonarthritic vehicle-treated group (81715.7±1984.1 vs 60269.9±2142.8).Indomethacin significantly decreased the serum TGF-β1 level after 3mg/kg/day p.o. (for 10 days) by about 1.5 times, compared to thevehicle-treated arthritic group (57222.2±3194.1 vs 81715.7±1984.1). E3(1 mg/kg i.v. on D 14 and D 19) and 911 (10 mg/kg i.v. on D 14 and D 19)significantly decreased the serum TGF-β1 level so that the cytokinelevel in E3- and 911-treated groups were comparable with those observedin vehicle-treated non arthritic group (69408.8±3926.7 and67214.5±3649.4 respectively, vs 60269.9±2142.8). TABLE 16 Effect of E3and 911 after i.v. injection (2 days on D 14 and D 19) on serum TGF-β1levels in rheumatoid arthritic rats Arthritic Rats Non-arthritic Rats E3911 Indomethacin vehicle i.v. vehicle i.v. 1 mg/kg i.v. 10 mg/kg i.v. 3mg/kg p.o. TGF-β1 60269.9 ± 2142.8 81715.7 ± 1984.1 69408.8 ± 3926.7 *67214.5 ± 3649.4 * 57222.2 ± 3194.1 *

[0520] Values are expressed in pg/ml, as Mean±S.E.M.

[0521] n=10 animals per group except for Non-arthritic/vehicle (Rightpaw), Arthritic/vehicle (Left paw) and Indomethacin (n=9)

[0522] Dunnett t test: * P≦50.05 vs vehicle-treated arthritic rats

[0523] 7. Hematological Parameters

[0524] As shown in Table 17, the hematological parameters such as whiteblood cells and platelets were greater in vehicle-treated arthritic ratsin comparison to vehicle-treated non arthritic rats (Student t testP<0.05), whereas the red blood cells, hemoglobin and hematocrit (Studentt test P>0.05) were unchanged. Indomethacin did not affect the bloodparameters after 3 mg/kg/day p.o. (for 10 days) compared to thevehicle-treated arthritic group. E3 (1 mg/kg i.v. on D 14 and D 19) didnot affect the blood parameters compared to the vehicle-treatedarthritic group. 911 (10 mg/kg i.v. on D 14 and D 19) did not affect theblood parameters compared to the vehicle-treated arthritic group. TABLE17 Effects of E3 and 911 after i.v. injection (2 days on D 14 and D 19)on blood parameters in rheumatoid arthritis in rats (Measurement at D24) White Red blood cells blood cells Hemoglobin Hematocrit PlateletsDay 10³/mm³ 10⁶/mm³ g/dl % 10³/mm³ Non vehicle i.v.  8.7 ± 0.9 7.98 ±0.31 15.1 ± 0.7 42.6 ± 1.6   322 ± 89 Arthritic n = 9 n = 9 n = 9 n = 9n = 9 Rats Arthritic vehicle i.v. 19.0 ± 0.9 7.54 ± 0.31 13.2 ± 0.7 37.4± 1.6 10.43 ± 89 Rats n = 10 n = 10 n = 10 n = 10 n = 10 E3 1 mg/kg i.v.19.1 ± 1.2 7.74 ± 0.17 12.9 ± 0.3 38.5 ± 1.0   827 ± 77 n = 7 n = 8 n =8 n = 8 n = 8 911 10 mg/kg i.v. 22.6 ± 2.9 7.30 ± 0.40 12.1 ± 0.7 36.5 ±2.1   799 ± 121 n = 8 n = 9 n = 9 n = 9 n = 9 Indomethacin 21.7 ± 2.56.93 ± 0.31 11.8 ± 0.6 35.0 ± 1.5   705 ± 111 3 mg/kg p.o. n = 9 n = 9 n= 9 n = 9 n = 9 over 10 days

[0525] Values are expressed as Mean±S.E.M.

[0526] Anova: P>0.05 vs vehicle-treated arthritic rats

[0527] 7. Hindpaw Weight

[0528] As shown in Table 18, the left and right hindpaw weight wasgreater in vehicle-treated arthritic rats than in vehicle-treated nonarthritic rats (3.43±0.11 vs 1.98±0.01 and 3.32±0.12 vs 1.99±0.02 g,respectively) (Student t test or Mann-Withney P<0.05). Indomethacinsignificantly decreased the hindpaws weight after 3 mg/kg/day p.o. (for10 days) compared to the vehicle-treated arthritic group (left hindpaw:2.23±0.04 vs 3.43±0.11 g; right hindpaw: 2.20±0.05 vs 3.32±0.12 g). E3(1 mg/kg i.v. on D 14 and D 19) only significantly increased the lefthindpaw weight compared to the vehicle-treated arthritic group (lefthindpaw: 3.86±0.14 vs 3.43±0.11 g; right hindpaw: 3.72±0.13 vs 3.32+0.12g). 911 (10 mg/kg i.v. on D 14 and D 19) only significantly increasedthe right hindpaw weight compared to the vehicle-treated arthritic group(left hindpaw: 3.73±0.12 vs 3.43±0.11 g; right hindpaw: 3.83±0.15 vs3.32±0.12 g). TABLE 18 Effects of E3 and 911 after i.v. injection (2days on D 14 and D 19) on hindpaws weight in rheumatoid arthritis inrats (Measurement at D 24) Left paw Right paw Non vehicle i.v 1.98 ±0.01 1.99 ± 0.02 Arthritic Rats Arthritic vehicle i.v. 3.43 ± 0.11 3.32± 0.12 Rats E3 1 mg/kg i.v. 3.86 ± 0.14 * 3.72 ± 0.13 911 10 mg/kg i.v.3.73 ± 0.12 3.83 ± 0.15 * Indomethacin 2.23 ± 0.04 * 2.20 ± 0.05 * 3mg/kg p.o. over 10 days

[0529] 8. X-ray Analysis

[0530] As shown in Table 19, a total score of 0.0±0.0 was observed inthe vehicle-treated non arthritic rats. The vehicle-treated arthriticrats have a total score of 15.1±1.3 with high scores fordemineralization (2.4±0.3), erosions (2.7±0.3), soft tissue damage(3.1±0.2) and space joint (3.3±0.2), a moderate score for periostalreaction (1.0±0.3), osteogenesis (0.8±0.2) and deformity (1.8±0.2).Indomethacin (3 mg/kg/day p.o. for 10 days) strongly and significantlydecreased the total score by about 10.7 in comparison to vehicle-treatedarthritic rats (4.4±0.9 vs 15.1±1.3). E3 (1 mg/kg i.v. on D 14 and D 19)did not affect the total score compared to the vehicle-treated arthriticgroup (14.2±1.3 vs 15.1±1.3). 911 (10 mg/kg i.v. on D 14 and D 19) didnot affect the total score compared to the vehicle-treated arthriticgroup (15.4±1.0 vs 15.1±1.3). TABLE 19 Effects of E3 and 911 after i.v.injection (2 days on D 14 and D 19) on X-ray parameters in rheumatoidarthritis in rats Soft Deminer- Periostal tissue Space osteo- TOTAL Dayalization Erosions reaction damage joint genesis Deformity score Nonvehicle i.v. 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.00.0 ± 0.0  0.0 ± 0.0 Arthritic Rats Arthritic vehicle i.v. 2.4 ± 0.3 2.7± 0.3 1.0 ± 0.3 3.1 ± 0.2 3.3 ± 0.2 0.8 ± 0.2 1.8 ± 0.2 15.1 ± 1.3 RatsE3 1 mg/kg i.v. 2.0 ± 0.2 2.4 ± 0.3 0.8 ± 0.2 3.3 ± 0.3 2.7 ± 0.2 1.2 ±0.2 1.8 ± 0.2 14.2 ± 1.3 911 10 mg/kg i.v. 2.3 ± 0.3 2.5 ± 0.2 1.0 ± 0.33.4 ± 0.2 3.3 ± 0.2 0.9 ± 0.2 2.0 ± 0.2 15.4 ± 1.0 Indomethacin 0.3 ±0.2 * 0.9 ± 0.2 * 0.7 ± 0.3 1.0 ± 0.2 * 1.0 ± 0.2 * 0.1 ± 0.1 0.4 ±0.2 *  4.4 ± 0.9 * 3 mg/kg p.o.

[0531] Values are expressed as Mean±S.E.M (score).

[0532] n=10 animals per group except for Indomethacin (n=9)

[0533] Dunnett t test: * P≦0.05 vs vehicle-treated arthritic rats

CONCLUSION

[0534] Under experimental conditions described above, E3 (1 mg/kg i.v. 2days: D 14-D 19) and 911 (10 mg/kg i.v. 2 days: D 14-D 19) showed stronganalgesic effects, but did not show significant anti-inflammatoryeffects in this arthritis model.

Example 9 Effects of Different Doses of Anti-NGF Antibody E3 in a RatModel of Rheumatoid Arthritis

[0535] The ability of E3 to produce reduction in pain in arthritic ratswas further investigated by examining the dose response relationshipbetween E3 administration and pain reduction. Rats were treated withadjuvant to induce arthritis as described above. Ten rats not injectedwith adjuvant were used as non-arthritic controls. Fourteen days afteradjuvant injection, animals were qualified into the study based on thecriteria stated above, randomized into eight groups of ten rats andtested for the intensity of their vocalization response. They were thendosed on day 14 with saline, or 0.003 mg/kg, 0.01 mg/kg, 0.03 mg/kg, 0.1mg/kg, 0.3 mg/kg, 1 mg/kg or 5 mg/kg of E3 antibody as described above.Animals were tested for their vocalization response on days 16, 18, 20,and 24. Animals were redosed with saline or the same dose of E3 on day18 after the vocalization testing. Animals were also weighed each day,starting at day 14. Thus, animals were dosed twice with a given dose ofantibody or saline on days 14 and 18, and assessed for pain five times,on days 14, 16, 18, 20 and 24. Data are shown in Tables 20-22 and inFIGS. 20-22. TABLE 20 Effects of different doses of E3 on nociceptiveresponse (vocalization intensity) in rheumatoid arthritic rats.Vocalization intensity values are expressed in mV as mean ± s.e.m. 0.0030.01 0.03 0.1 0.3 1.0 5.0 vehicle mg/kg mg/kg mg/kg mg/kg mg/kg mg/kgmg/kg day 14 mean 1129.25 981.75 1007.28 963.18 1159.30 1191.58 1067.00896.25 s.e.m 143.06 71.00 66.50 62.12 132.76 123.44 69.73 57.53 day 16mean 1042.85 825.60 576.88 448.43 283.71 151.85 98.62 79.18 s.e.m 130.5157.94 49.71 81.01 60.00 26.08 29.17 27.30 day 18 mean 968.10 427.43334.45 292.52 262.96 194.19 174.13 200.42 s.e.m 117.85 48.55 35.10 52.3662.32 53.56 88.61 120.15 day 20 mean 942.18 448.00 313.13 209.48 79.7466.27 71.23 63.57 s.e.m 100.69 33.73 61.98 24.43 33.18 31.34 42.37 23.47day 24 mean 913.68 724.50 596.38 513.60 432.45 176.32 19.21 12.35 s.e.m131.29 115.90 44.76 63.67 70.38 66.61 10.14 12.35

[0536] The effect of treating animals with various doses of anti-NGFantibody E3 on pain induced vocalization (data shown in Table 20) wasstatistically analyzed by using two-way ANOVA to compare the resultsobtained pairwise between arthritic animals treated with vehicle withthose treated with a given dose of antibody E3. There was a highlysignificant effect at all levels of E3 tested (p<0.0001). Even at thelowest dose tested (0.003 mg/kg of E3), the difference in vocalizationwas significant (p<0.0001).

[0537] As shown in Table 20 and FIG. 20, in agreement with the aboveexperiments, treatment with antibody E3 at 1 mg/kg showed a rapid androbust relief of pain. Within two days (the earliest time point tested)the vocalization intensity fell by 90%. Treatment with lowerconcentrations of E3 also provided robust pain relief, although at lowerdoses the pain relief took somewhat longer to manifest. It is likelythat the apparent decrease in efficacy on day 24 of all but the highestdoses tested is due to a decrease in the actual level of plasma E3 levelsecondary to an immune response by the subject rats. It is apparent thatdoses as low as 0.003 mg/kg provide at least partial pain relief in thismodel. TABLE 21 Effects of different doses of E3 on body weight inrheumatoid arthritic rats (normalized to day 14). Non-Arthritic vehicle0.003 mg/kg 0.01 mg/kg 0.03 mg/kg Day Mean S.E.M Mean S.E.M Mean S.E.MMean S.E.M Mean S.E.M 14 100.00 0.00 100.00 0.00 100.00 0.00 100.00 0.00100.00 0.00 15 99.53 0.30 99.14 0.37 99.20 0.48 99.18 0.43 100.34 0.3616 102.52 0.45 99.57 0.60 99.58 0.79 99.33 0.72 100.89 0.57 17 103.310.41 99.50 0.64 100.46 0.77 99.69 0.73 101.80 0.82 18 106.11 0.72 100.260.93 100.90 1.19 100.69 0.72 102.70 0.92 20 109.62 0.85 101.46 1.22102.26 1.58 102.70 1.07 104.51 0.75 21 110.52 0.93 102.73 1.49 103.161.87 102.63 1.18 105.08 0.98 23 114.28 1.19 104.54 1.92 106.09 1.67104.41 1.33 106.14 1.06 24 115.44 1.15 105.12 1.92 106.16 1.90 104.231.46 106.23 1.26 0.1 mg/kg 0.3 mg/kg 1.0 mg/kg 5.0 mg/kg Day Mean S.E.MMean S.E.M Mean S.E.M Mean S.E.M 14 100.00 0.00 100.00 0.00 100.00 0.00100.00 0.00 15 99.83 0.59 101.05 0.38 100.53 0.37 101.61 0.41 16 101.070.82 102.88 0.50 102.95 0.56 104.09 0.60 17 101.89 1.12 104.76 0.70105.74 0.76 106.85 0.79 18 103.69 1.47 107.11 0.78 108.46 0.82 109.531.00 20 107.36 1.78 111.26 0.77 113.57 0.83 115.32 1.11 21 108.50 2.01113.31 0.87 116.71 0.92 119.11 1.21 23 109.25 2.15 115.59 1.38 123.351.13 126.36 1.94 24 108.77 2.08 115.58 1.43 124.41 1.00 127.25 1.79

[0538] TABLE 22 Effects of different doses of E3 on body weight inrheumatoid arthritic rats (normalized to day 0). Non-Arthritic vehicle0.003 mg/kg 0.01 mg/kg 0.03 mg/kg Day Mean S.E.M Mean S.E.M Mean S.E.MMean S.E.M Mean S.E.M 0 100.00 0.00 100.00 0.00 100.00 0.00 100.00 0.00100.00 0.00 1 100.45 0.19 98.34 0.48 98.37 0.35 98.86 0.33 98.67 0.34 2105.94 0.33 101.75 0.71 102.47 0.59 102.61 0.40 102.05 0.53 3 109.290.33 105.04 1.04 106.54 0.99 106.29 0.60 105.31 0.85 4 113.13 0.46109.14 1.15 110.09 0.72 110.61 0.41 109.24 0.82 7 124.15 0.70 119.901.39 121.29 1.32 121.59 0.72 117.15 1.36 8 127.82 0.80 123.38 1.52124.44 1.43 124.47 1.24 118.52 1.89 9 132.40 0.80 125.50 1.59 125.911.69 125.82 1.95 118.60 2.62 10 135.91 0.83 123.51 1.77 123.30 2.47123.87 2.59 115.26 3.19 11 140.42 1.13 119.82 1.98 119.55 2.76 121.202.99 112.94 3.48 14 152.59 1.72 111.79 1.40 111.50 1.87 111.80 1.65108.37 2.75 15 151.87 1.87 110.82 1.41 110.63 2.05 110.85 1.44 108.682.45 16 156.47 2.25 111.33 1.74 111.08 2.32 110.98 1.31 109.21 2.16 17157.65 2.08 111.24 1.62 112.06 2.36 111.42 1.66 110.16 2.03 18 161.982.71 112.16 2.21 112.60 2.78 112.54 1.64 111.14 2.11 20 167.36 2.93113.49 2.37 114.17 3.24 114.82 2.12 113.17 2.49 21 168.73 3.07 114.932.62 115.25 3.68 114.76 2.30 113.80 2.68 23 174.51 3.54 116.96 3.02118.48 3.49 116.76 2.51 114.93 2.62 24 176.27 3.50 117.63 3.13 118.583.71 116.56 2.57 114.99 2.51 0.1 mg/kg 0.3 mg/kg 1.0 mg/kg 5.0 mg/kg DayMean S.E.M Mean S.E.M Mean S.E.M Mean S.E.M 0 100.00 0.00 100.00 0.00100.00 0.00 100.00 0.00 1 99.31 0.61 99.26 0.28 98.81 0.27 98.25 0.58 2102.87 0.73 102.98 0.43 103.18 0.50 101.82 0.53 3 106.26 0.82 106.950.50 106.52 0.55 105.47 0.58 4 110.20 0.64 110.50 0.58 110.52 0.67109.29 0.58 7 120.50 1.20 120.03 0.82 121.54 1.15 119.77 1.19 8 123.481.58 121.38 1.31 124.28 1.59 121.96 1.72 9 125.46 2.47 121.57 2.09125.60 2.23 123.04 2.42 10 123.95 3.38 118.27 3.07 124.11 2.97 120.002.81 11 121.98 3.93 116.02 3.32 121.27 3.42 117.97 2.98 14 113.90 2.14108.43 1.94 111.72 2.27 111.58 2.59 15 113.66 1.91 109.59 2.12 112.302.23 113.33 2.37 16 115.06 2.00 111.54 2.02 115.00 2.36 116.06 2.30 17115.99 2.18 113.57 2.04 118.08 2.32 119.14 2.42 18 118.01 2.29 116.132.14 121.16 2.55 122.14 2.61 20 122.17 2.57 120.62 2.20 126.90 2.87128.60 2.77 21 123.49 2.90 122.88 2.49 130.41 2.98 132.82 2.84 23 124.353.02 125.36 2.83 137.81 3.09 140.79 2.83 24 123.77 2.80 125.33 2.75138.93 2.76 141.77 2.61

[0539] The effect of treating animals with various doses of anti-NGFantibody E3 on eight was statistically analyzed by using two-way ANOVAto compare the results obtained pairwise between arthritic animalstreated with vehicle with those treated with a given dose of antibodyE3. Using data normalized to weight on day 14 (Table 21), doses of 0.03mg/kg of E3 resulted in a significant change in body weight (p<0.005).At all higher dose of E3, the difference between treated and untreatedarthritic animals was significant (p=or <0.0001). Using data normalizedto weight on day 0 (Table 22), dose of 0.03 mg/kg of E3 resulted in asignificant change in body weight (p<0.002). At all higher dose of E3,the difference between treated and untreated arthritic animals wassignificant (p<0.0001).

[0540] Again in agreement with earlier studies, rats treated with E3showed less apparent weight loss than saline treated arthritic rats(Table 22 and FIG. 22). In fact, rats treated with high doses ofantibody E3 were recovering the earlier weight loss, and actuallygaining weight faster than their non-arthritic cohorts (Table 21 andFIG. 21).

[0541] Deposit of Biological Material

[0542] The following materials have been deposited with the AmericanType Culture Collection, 10801 University Boulevard, Manassas, Va., USA(ATCC): ATCC Material Accession No. Date of Deposit Eb.911.3E E3 lightchain PTA-4893 Jan. 8, 2003 Eb.pur.911.3E E3 light chain PTA-4894 Jan.8, 2003 Db.911.3E E3 heavy chain PTA-4895 Jan. 8, 2003

[0543] Vector Eb.911.3E is a polynucleotide encoding the E3 light chainvariable region; vector Eb.pur.911.3E is a polynucleotide encoding E3light chain variable region, and vector Db.911.3E is a polynucleotideencoding the E3 heavy chain variable region.

[0544] This deposit was made under the provisions of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe Purpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit. The deposit will be made available byATCC under the terms of the Budapest Treaty, and subject to an agreementbetween Rinat Neuroscience Corp. and ATCC, which assures permanent andunrestricted availability of the progeny of the culture of the depositto the public upon issuance of the pertinent U.S. patent or upon layingopen to the public of any U.S. or foreign patent application, whichevercomes first, and assures availability of the progeny to one determinedby the U.S. Commissioner of Patents and Trademarks to be entitledthereto according to 35 USC Section 122 and the Commissioner's rulespursuant thereto (including 37 CFR Section 1.14 with particularreference to 8860G 638).

[0545] The assignee of the present application has agreed that if aculture of the materials on deposit should die or be lost or destroyedwhen cultivated under suitable conditions, the materials will bepromptly replaced on notification with another of the same. Availabilityof the deposited material is not to be construed as a license topractice the invention in contravention of the rights granted under theauthority of any government in accordance with its patent laws. Antibodysequences Heavy chain variable region (Kabat CDRs are underlined;Chothia CDRs are

) QVQLQESGPGLVKPSETLSLTCTVSGFSLI

WIRQPPGKGLEWIG

(SEQ ID NO:1)

RVTISKDTSKNQFSLKLSSVTAADTAVYYCAR

WGQGTL VTVS Light chain variable region (Kabat CDRs are underlined;Chothia CDRs are

) DIQMTQSPSSLSASVGDRVTITC

WYQQKPGKAPKLLIY

GVP (SEQ ID NO:2) SRFSGSGSGTDFTFTISSLQPEDIATYYC

FGQGTKLEIKRT E3 heavy chain extended CDRs CDRH1: GFSLIGYDLN (SEQ IDNO:3) CDRH2: IIWGDGTTDYNSAVKS (SEQ ID NO:4) CDRH3: GGYWYATSYYFDY (SEQ IDNO:5) E3 light chain extended CDRs CDRL1: RASQSISNNLN (SEQ ID NO:6)CDRL2: YTSRFHS (SEQ ID NO:7) CDRL3: QQEHTLPYT (SEQ ID NO:8) Mousemonoclonal antibody 911 extended CDRs 911 heavy chain extended CDRsCDRH1: GFSLIGYDIN (SEQ ID NO:9) CDRH2: MIWGDGTTDYNSALKS (SEQ ID NO:10)CDRH3: GGYYYGTSYYFDY (SEQ ID NO:11) 911 light chain extended CDRs CDRL1:RASQDISNHLN (SEQ ID NO:12) CDRL2: YISRFHS (SEQ ID NO:13) CDRL3:QQSKTLPYT (SEQ ID NO:14) E3 heavy chain amino acid sequence (full)QVQLQESGPGLVKPSETLSLTCTVSGFSLIGYDLNWIRQPPGKGLEWIGIIWGDGTTD (SEQ IDNO:16) YNSAVKSRVTISKDTSKNQFSLKLSSVTAADTAVYYCARGGYWYATSYYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 3E light chain aminoacid sequence (full antibody)DIQMTQSPSSLSASVGDRVTITCRASQSISNNLNWYQQKPGKAPKLLIYYTSRFHSGV (SEQ IDNO:17) PSRFSGSGSGTDFTFTISSLQPEDIATYYCQQEHTLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHXVYACEVTHQGLSSPVTKSFNRGEC 3E heavy chain nucleotidesequence (full antibody)CAGGTGCAGCTGCAGGAGTCTGGCCCAGGACTGGTGAAGCCTTCCGAGACCCTGT (SEQ ID NO: 65)CCCTCACCTGCACTGTCTCTGGGTTCTCACTTATCGGCTATGATCTTAACTGGATCCGACAGCCTCCAGGGAAGGGACTGGAGTGGATTGGGATTATCTGGGGTGATGGAACCACAGACTATAATTCAGCTGTCAAATCCCGCGTCACCATCTCAAAAGACACCTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCCGTGTATTACTGTGCGAGAGGAGGTTATTGGTACGCCACTAGCTACTACTTTGACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCTGTCTTCCCACTGGCCCCATGCTCCCGCAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCAGAACCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTGCAGTCCTCAGGTCTCTACTCCCTCAGCAGCGTGGTGACCGTGCCATCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCAAGCAACACCAAGGTCGACAAGACCGTGGAGAGAAAGTGTTGTGTGGAGTGTCCACCTTGTCCAGCCCCTCCAGTGGCCGGACCATCCGTGTTCCTGTTCCCTCCAAAGCCAAAGGACACCCTGATGATCTCCAGAACCCCAGAGGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCAGAGGTGCAGTTCAACTGGTATGTGGACGGAGTGGAGGTGCACAACGCCAAGACCAAGCCAAGAGAGGAGCAGTTCAACTCCACCTTCAGAGTGGTGAGCGTGCTGACCGTGGTGCACCAGGACTGGCTGAACGGAAAGGAGTATAAGTGTAAGGTGTCCAACAAGGGACTGCCATCCAGCATCGAGAAGACCATCTCCAAGACCAAGGGACAGCCAAGAGAGCCACAGGTGTATACCCTGCCACCATCCAGAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGATTCTATCCATCCGACATCGCCGTGGAGTGGGAGTCCAACGGACAGCCAGAGAACAACTATAAGACCACCCCTCCAATGCTGGACTCCGACGGATCCTTCTTCCTGTATTCCAAGCTGACCGTGGACAAGTCCAGATGGCAGCAGGGAAACGTGTTCTCTTGTTCCGTGATGCACGAGGCCCTGCACAACCACTATACCCAGAAGAGCCTGTCCCTGTC TCCAGGAAAGTAA3E heavy chain variable domain nucleotide sequenceCAGGTGCAGCTGCAGGAGTCTGGCCCAGGACTGGTGAAGCCTTCCGAGACCCTGT (SEQ ID NO:66)CCCTCACCTGCACTGTCTCTGGGTTCTCACTTATCGGCTATGATCTTAACTGGATCCGACAGCCTCCAGGGAAGGGACTGGAGTGGATTGGGATTATCTGGGGTGATGGAACCACAGACTATAATTCAGCTGTCAAATCCCGCGTCACCATCTCAAAAGACACCTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCCGTGTATTACTGTGCGAGAGGAGGTTATTGGTACGCCACTAGCTACTACTTTGACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA 3E light chain nucleotide sequence (fullantibody) GATATCCAGATGACACAGTCCCCATCCTCCCTGTCTGCCTCTGTGGGTGACCGCGT (SEQID NO:67) CACCATCACCTGCCGCGCATCTCAGTCCATTAGCAATAATCTGAACTGGTATCAGCAGAAGCCAGGCAAAGCCCCAAAACTCCTGATCTACTACACCTCACGCTTCCACTCAGGTGTCCCATCACGCTTCAGTGGCAGTGGCTCTGGTACAGATTTCACCTTCACCATTAGCAGCCTGCAACCAGAAGATATTGCCACTTATTACTGCCAACAGGAGCATACCCTTCCATATACCTTCGGTCAAGGCACCAAGCTGGAGATCAAACGCACTGTGGCTGCACCATCTGTCTTCATCTTTCCTCCATCTGATGAGCAGTTGAAATCCGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCACGCGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCCGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACCCTGAGCAAAGCAGACTACGAGAAACACMAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCTCCAGTCACAAAGAGCTTCAACCGCGGTGAGTGCTAA 3E light chain variable domainnucleotide sequenceGATATCCAGATGACACAGTCCCCATCCTCCCTGTCTGCCTCTGTGGGTGACCGCGT (SEQ ID NO:68)CACCATCACCTGCCGCGCATCTCAGTCCATTAGCAATAATCTGAACTGGTATCAGCAGAAGCCAGGCAAAGCCCCAAAACTCCTGATCTACTACACCTCACGCTTCCACTCAGGTGTCCCATCACGCTTCAGTGGCAGTGGCTCTGGTACAGATTTCACCTTCACCATTAGCAGCCTGCAACCAGAAGATATTGCCACTTATTACTGCCAACAGGAGCATACCCTTCCATATACCTTCGGTCAAGGCACCAAGCTGGAGATCAAACGC

[0546] It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication.

1 68 1 120 PRT Artificial Sequence Synthetic Construct 1 Gln Val Gln LeuGln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu SerLeu Thr Cys Thr Val Ser Gly Phe Ser Leu Ile Gly Tyr 20 25 30 Asp Leu AsnTrp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Ile IleTrp Gly Asp Gly Thr Thr Asp Tyr Asn Ser Ala Val Lys 50 55 60 Ser Arg ValThr Ile Ser Lys Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys LeuSer Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg GlyGly Tyr Trp Tyr Ala Thr Ser Tyr Tyr Phe Asp Tyr Trp Gly 100 105 110 GlnGly Thr Leu Val Thr Val Ser 115 120 2 109 PRT Artificial SequenceSynthetic Construct 2 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu SerAla Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser GlnSer Ile Ser Asn Asn 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys AlaPro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Phe His Ser Gly Val ProSer Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr IleSer Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln GlnGlu His Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu IleLys Arg Thr 100 105 3 10 PRT Artificial Sequence Synthetic Construct 3Gly Phe Ser Leu Ile Gly Tyr Asp Leu Asn 1 5 10 4 16 PRT ArtificialSequence Synthetic Construct 4 Ile Ile Trp Gly Asp Gly Thr Thr Asp TyrAsn Ser Ala Val Lys Ser 1 5 10 15 5 13 PRT Artificial Sequence SyntheticConstruct 5 Gly Gly Tyr Trp Tyr Ala Thr Ser Tyr Tyr Phe Asp Tyr 1 5 10 611 PRT Artificial Sequence Synthetic Construct 6 Arg Ala Ser Gln Ser IleSer Asn Asn Leu Asn 1 5 10 7 7 PRT Artificial Sequence SyntheticConstruct 7 Tyr Thr Ser Arg Phe His Ser 1 5 8 9 PRT Artificial SequenceSynthetic Construct 8 Gln Gln Glu His Thr Leu Pro Tyr Thr 1 5 9 10 PRTArtificial Sequence Synthetic Construct 9 Gly Phe Ser Leu Ile Gly TyrAsp Ile Asn 1 5 10 10 16 PRT Artificial Sequence Synthetic Construct 10Met Ile Trp Gly Asp Gly Thr Thr Asp Tyr Asn Ser Ala Leu Lys Ser 1 5 1015 11 13 PRT Artificial Sequence Synthetic Construct 11 Gly Gly Tyr TyrTyr Gly Thr Ser Tyr Tyr Phe Asp Tyr 1 5 10 12 11 PRT Artificial SequenceSynthetic Construct 12 Arg Ala Ser Gln Asp Ile Ser Asn His Leu Asn 1 510 13 7 PRT Artificial Sequence Synthetic Construct 13 Tyr Ile Ser ArgPhe His Ser 1 5 14 9 PRT Artificial Sequence Synthetic Construct 14 GlnGln Ser Lys Thr Leu Pro Tyr Thr 1 5 15 15 PRT Artificial SequenceSynthetic Construct 15 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly GlyGly Gly Ser 1 5 10 15 16 447 PRT Artificial Sequence Synthetic Construct16 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 510 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ile Gly Tyr 2025 30 Asp Leu Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 3540 45 Gly Ile Ile Trp Gly Asp Gly Thr Thr Asp Tyr Asn Ser Ala Val Lys 5055 60 Ser Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Phe Ser Leu 6570 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala85 90 95 Arg Gly Gly Tyr Trp Tyr Ala Thr Ser Tyr Tyr Phe Asp Tyr Trp Gly100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly ProSer 115 120 125 Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu SerThr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu ProVal Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly ValHis Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser LeuSer Ser Val Val Thr Val 180 185 190 Pro Ser Ser Asn Phe Gly Thr Gln ThrTyr Thr Cys Asn Val Asp His 195 200 205 Lys Pro Ser Asn Thr Lys Val AspLys Thr Val Glu Arg Lys Cys Cys 210 215 220 Val Glu Cys Pro Pro Cys ProAla Pro Pro Val Ala Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro ProLys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val ThrCys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Gln PheAsn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr LysPro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser 290 295 300 ValLeu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315320 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 325330 335 Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr CysLeu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp GluSer Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro MetLeu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu ThrVal Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys SerVal Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser LeuSer Leu Ser Pro Gly Lys 435 440 445 17 214 PRT Artificial SequenceSynthetic Construct 17 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu SerAla Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser GlnSer Ile Ser Asn Asn 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys AlaPro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Phe His Ser Gly Val ProSer Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr IleSer Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln GlnGlu His Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu IleLys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro SerAsp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu LeuAsn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val AspAsn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr GluGln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu ThrLeu Ser Lys Ala Asp Tyr Glu Lys His Xaa Val Tyr 180 185 190 Ala Cys GluVal Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe AsnArg Gly Glu Cys 210 18 11 PRT Artificial Sequence Synthetic Construct 18Arg Ala Ser Gln Ser Ile Ser Asn Asn Leu Asn 1 5 10 19 7 PRT ArtificialSequence Synthetic Construct 19 Tyr Thr Ser Arg Phe His Ser 1 5 20 11PRT Artificial Sequence Synthetic Construct 20 Arg Ala Ser Gln Tyr IleSer Asn His Leu Asn 1 5 10 21 7 PRT Artificial Sequence SyntheticConstruct 21 Tyr Thr Ser Arg Phe His Ser 1 5 22 11 PRT ArtificialSequence Synthetic Construct 22 Arg Ala Ser Gln Ser Ile Ser Asn Gln LeuAsn 1 5 10 23 7 PRT Artificial Sequence Synthetic Construct 23 Tyr ValSer Arg Phe His Ser 1 5 24 11 PRT Artificial Sequence SyntheticConstruct 24 Arg Ala Phe Gln Ala Ile Ser Asn Gln Leu Asn 1 5 10 25 7 PRTArtificial Sequence Synthetic Construct 25 Tyr Ile Ser Arg Phe His Thr 15 26 11 PRT Artificial Sequence Synthetic Construct 26 Arg Ala Phe GlnSer Ile Ser Asn Gln Leu Asn 1 5 10 27 7 PRT Artificial SequenceSynthetic Construct 27 Tyr Ala Ser Arg Phe His Ser 1 5 28 10 PRTArtificial Sequence Synthetic Construct 28 Gly Phe Ser Leu Ile Gly TyrAsp Ser Asn 1 5 10 29 14 PRT Artificial Sequence Synthetic Construct 29Ile Ile Trp Gly Asp Gly Thr Thr Asp Tyr Asn Ser Ala Leu 1 5 10 30 10 PRTArtificial Sequence Synthetic Construct 30 Gly Phe Ser Leu Ile Gly TyrAsp Leu Asn 1 5 10 31 14 PRT Artificial Sequence Synthetic Construct 31Ile Ile Trp Gly Asp Gly Thr Thr Asp Tyr Asn Ser Ala Val 1 5 10 32 10 PRTArtificial Sequence Synthetic Construct 32 Gly Phe Ser Leu Ile Gly TyrAsp Val Thr 1 5 10 33 14 PRT Artificial Sequence Synthetic Construct 33Gly Ile Trp Gly Asp Gly Thr Thr Asp Tyr Asn Ser Ala Val 1 5 10 34 10 PRTArtificial Sequence Synthetic Construct 34 Gly Phe Ser Leu Ile Gly TyrAsp Val Thr 1 5 10 35 14 PRT Artificial Sequence Synthetic Construct 35Gly Ile Trp Gly Asp Gly Thr Thr Asp Tyr Asn Ser Ser Val 1 5 10 36 10 PRTArtificial Sequence Synthetic Construct 36 Gly Phe Ser Leu Ile Gly TyrAsp Ala Thr 1 5 10 37 14 PRT Artificial Sequence Synthetic Construct 37Gly Ile Trp Gly Asp Gly Thr Thr Asp Tyr Asn Ser Ala Val 1 5 10 38 10 PRTArtificial Sequence Synthetic Construct 38 Gly Phe Ser Leu Ile Gly TyrAsp Val Ser 1 5 10 39 14 PRT Artificial Sequence Synthetic Construct 39Ile Ile Trp Gly Asp Gly Thr Thr Asp Tyr Asn Ser Ser Val 1 5 10 40 10 PRTArtificial Sequence Synthetic Construct 40 Gly Phe Ser Leu Ile Gly TyrAsp Ile Ser 1 5 10 41 14 PRT Artificial Sequence Synthetic Construct 41Gln Ile Trp Gly Asp Gly Thr Thr Asp Tyr Asn Ser Ser Val 1 5 10 42 10 PRTArtificial Sequence Synthetic Construct 42 Gly Phe Ser Leu Ile Gly TyrAsp Ala Ser 1 5 10 43 14 PRT Artificial Sequence Synthetic Construct 43Gly Ile Trp Gly Asp Gly Thr Thr Asp Tyr Asn Ser Ser Val 1 5 10 44 10 PRTArtificial Sequence Synthetic Construct 44 Gly Phe Ser Leu Ile Gly TyrAsp Ser Thr 1 5 10 45 14 PRT Artificial Sequence Synthetic Construct 45Ser Ile Trp Gly Asp Gly Thr Thr Asp Tyr Asn Ser Ala Leu 1 5 10 46 13 PRTArtificial Sequence Synthetic Construct 46 Gly Gly Tyr Trp Tyr Gly ThrSer Tyr Tyr Phe Asp Tyr 1 5 10 47 13 PRT Artificial Sequence SyntheticConstruct 47 Gly Gly Tyr Tyr Tyr Gly Thr Ala Tyr Tyr Phe Asp Tyr 1 5 1048 13 PRT Artificial Sequence Synthetic Construct 48 Gly Gly Tyr Tyr TyrGly Thr Thr Tyr Tyr Phe Asp Tyr 1 5 10 49 13 PRT Artificial SequenceSynthetic Construct 49 Gly Gly Tyr Tyr Tyr Ala Thr Ser Tyr Tyr Phe AspTyr 1 5 10 50 9 PRT Artificial Sequence Synthetic Construct 50 Gln GlnGlu Lys Thr Leu Pro Tyr Thr 1 5 51 9 PRT Artificial Sequence SyntheticConstruct 51 Gln Gln Glu Ala Thr Leu Pro Tyr Thr 1 5 52 13 PRTArtificial Sequence Synthetic Construct 52 Gly Gly Tyr Trp Tyr Ala ThrSer Tyr Tyr Phe Asp Tyr 1 5 10 53 9 PRT Artificial Sequence SyntheticConstruct 53 Gln Gln Glu Arg Thr Leu Pro Tyr Thr 1 5 54 13 PRTArtificial Sequence Synthetic Construct 54 Gly Gly Tyr Trp Tyr Ala ThrSer Tyr Tyr Phe Asp Tyr 1 5 10 55 9 PRT Artificial Sequence SyntheticConstruct 55 Gln Gln Glu His Thr Leu Pro Tyr Thr 1 5 56 13 PRTArtificial Sequence Synthetic Construct 56 Gly Gly Tyr Trp Tyr Ala ThrSer Tyr Tyr Phe Asp Tyr 1 5 10 57 9 PRT Artificial Sequence SyntheticConstruct 57 Gln Gln Glu Ser Thr Leu Pro Tyr Thr 1 5 58 13 PRTArtificial Sequence Synthetic Construct 58 Gly Gly Tyr Trp Tyr Ser ThrSer Tyr Tyr Phe Asp Tyr 1 5 10 59 9 PRT Artificial Sequence SyntheticConstruct 59 Gln Gln Glu Lys Thr Leu Pro Tyr Thr 1 5 60 13 PRTArtificial Sequence Synthetic Construct 60 Gly Gly Tyr Tyr Tyr Ala ThrSer Tyr Tyr Phe Asp Tyr 1 5 10 61 9 PRT Artificial Sequence SyntheticConstruct 61 Gln Gln Glu Arg Thr Leu Pro Tyr Thr 1 5 62 13 PRTArtificial Sequence Synthetic Construct 62 Gly Gly Tyr Trp Tyr Ala ThrSer Tyr Tyr Phe Asp Tyr 1 5 10 63 9 PRT Artificial Sequence SyntheticConstruct 63 Gln Gln Glu Arg Thr Leu Pro Tyr Thr 1 5 64 13 PRTArtificial Sequence Synthetic Construct 64 Gly Gly Tyr Tyr Tyr Ala ThrSer Tyr Tyr Phe Asp Tyr 1 5 10 65 1344 DNA Artificial Sequence SyntheticConstruct 65 caggtgcagc tgcaggagtc tggcccagga ctggtgaagc cttccgagaccctgtccctc 60 acctgcactg tctctgggtt ctcacttatc ggctatgatc ttaactggatccgacagcct 120 ccagggaagg gactggagtg gattgggatt atctggggtg atggaaccacagactataat 180 tcagctgtca aatcccgcgt caccatctca aaagacacct ccaagaaccagttctccctg 240 aagctgagct ctgtgaccgc cgcggacacg gccgtgtatt actgtgcgagaggaggttat 300 tggtacgcca ctagctacta ctttgactac tggggccagg gcaccctggtcaccgtctcc 360 tcagcctcca ccaagggccc atctgtcttc ccactggccc catgctcccgcagcacctcc 420 gagagcacag ccgccctggg ctgcctggtc aaggactact tcccagaacctgtgaccgtg 480 tcctggaact ctggcgctct gaccagcggc gtgcacacct tcccagctgtcctgcagtcc 540 tcaggtctct actccctcag cagcgtggtg accgtgccat ccagcaacttcggcacccag 600 acctacacct gcaacgtaga tcacaagcca agcaacacca aggtcgacaagaccgtggag 660 agaaagtgtt gtgtggagtg tccaccttgt ccagcccctc cagtggccggaccatccgtg 720 ttcctgttcc ctccaaagcc aaaggacacc ctgatgatct ccagaaccccagaggtgacc 780 tgtgtggtgg tggacgtgtc ccacgaggac ccagaggtgc agttcaactggtatgtggac 840 ggagtggagg tgcacaacgc caagaccaag ccaagagagg agcagttcaactccaccttc 900 agagtggtga gcgtgctgac cgtggtgcac caggactggc tgaacggaaaggagtataag 960 tgtaaggtgt ccaacaaggg actgccatcc agcatcgaga agaccatctccaagaccaag 1020 ggacagccaa gagagccaca ggtgtatacc ctgccaccat ccagagaggagatgaccaag 1080 aaccaggtgt ccctgacctg tctggtgaag ggattctatc catccgacatcgccgtggag 1140 tgggagtcca acggacagcc agagaacaac tataagacca cccctccaatgctggactcc 1200 gacggatcct tcttcctgta ttccaagctg accgtggaca agtccagatggcagcaggga 1260 aacgtgttct cttgttccgt gatgcacgag gccctgcaca accactatacccagaagagc 1320 ctgtccctgt ctccaggaaa gtaa 1344 66 363 DNA ArtificialSequence Synthetic Construct 66 caggtgcagc tgcaggagtc tggcccaggactggtgaagc cttccgagac cctgtccctc 60 acctgcactg tctctgggtt ctcacttatcggctatgatc ttaactggat ccgacagcct 120 ccagggaagg gactggagtg gattgggattatctggggtg atggaaccac agactataat 180 tcagctgtca aatcccgcgt caccatctcaaaagacacct ccaagaacca gttctccctg 240 aagctgagct ctgtgaccgc cgcggacacggccgtgtatt actgtgcgag aggaggttat 300 tggtacgcca ctagctacta ctttgactactggggccagg gcaccctggt caccgtctcc 360 tca 363 67 645 DNA ArtificialSequence Synthetic Construct 67 gatatccaga tgacacagtc cccatcctccctgtctgcct ctgtgggtga ccgcgtcacc 60 atcacctgcc gcgcatctca gtccattagcaataatctga actggtatca gcagaagcca 120 ggcaaagccc caaaactcct gatctactacacctcacgct tccactcagg tgtcccatca 180 cgcttcagtg gcagtggctc tggtacagatttcaccttca ccattagcag cctgcaacca 240 gaagatattg ccacttatta ctgccaacaggagcataccc ttccatatac cttcggtcaa 300 ggcaccaagc tggagatcaa acgcactgtggctgcaccat ctgtcttcat ctttcctcca 360 tctgatgagc agttgaaatc cggaactgcctctgttgtgt gcctgctgaa taacttctat 420 ccacgcgagg ccaaagtaca gtggaaggtggataacgccc tccaatccgg taactcccag 480 gagagtgtca cagagcagga cagcaaggacagcacctaca gcctcagcag caccctgacc 540 ctgagcaaag cagactacga gaaacacmaagtctacgcct gcgaagtcac ccatcagggc 600 ctgagttctc cagtcacaaa gagcttcaaccgcggtgagt gctaa 645 68 324 DNA Artificial Sequence Synthetic Construct68 gatatccaga tgacacagtc cccatcctcc ctgtctgcct ctgtgggtga ccgcgtcacc 60atcacctgcc gcgcatctca gtccattagc aataatctga actggtatca gcagaagcca 120ggcaaagccc caaaactcct gatctactac acctcacgct tccactcagg tgtcccatca 180cgcttcagtg gcagtggctc tggtacagat ttcaccttca ccattagcag cctgcaacca 240gaagatattg ccacttatta ctgccaacag gagcataccc ttccatatac cttcggtcaa 300ggcaccaagc tggagatcaa acgc 324

The following is claimed:
 1. An anti-nerve growth factor (NGF) antibodywherein said antibody: (a) binds NGF with a K_(D) of less than about 2nM; (b) inhibits human NGF-dependent survival of mouse E13.5 trigeminalneurons with an IC50 of about 100 pM or less, wherein the IC50 ismeasured in the presence of about 15 pM human NGF; and (c) inhibitshuman NGF-dependent survival of mouse E13.5 trigeminal neurons with anIC50 of about 10 pM or less, wherein the IC50 is measured in thepresence of about 1.5 pM of NGF.
 2. The antibody of claim 1, wherein theantibody is humanized.
 3. The antibody of claim 1, wherein the antibodyis affinity matured.
 4. The antibody of claim 1, wherein the antibody isa monoclonal antibody.
 5. The antibody of claim 1, wherein the antibodyis isolated.
 6. An antibody comprising a heavy chain variable regioncomprising: (a) a CDR1 region of SEQ ID NO:9, wherein I34 is S, L, V A,or I; and N35 is substituted with N, T or S; (b) a CDR2 region of SEQ IDNO:10, wherein M50 is I, G, Q, S, or L; A62 is A, or S; and L63 is L orV; and (c) a CDR3 region of SEQ ID NO:11, wherein Y100 is Y, L, or R;wherein Y101 is Y or W; wherein G103 is G, A, or S; wherein T104 is T orS; wherein S105 is S, A, or T; wherein Y106 is Y, R, T, or M; whereinY107 is Y or F; wherein F108 is F or W; wherein D109 is D, N, or G; andwherein Y110 is Y, K, S, R or T; wherein the antibody binds NGF.
 7. Theantibody of claim 6, wherein the antibody further comprises a lightchain variable region.
 8. An antibody comprising a light chain variableregion comprising: (a) a CDR1 region of SEQ ID NO:12, wherein S26 is Sor F; D28 is D, S, A, or Y; and H32 is H, N, or Q; (b) a CDR2 region ofSEQ ID NO:13, wherein I51 is I, T, V or A; and S56 is S or T; and (c) aCDR3 region of SEQ ID NO:14, wherein K92 is K, H, R, or S; and whereinY96 is Y or R; wherein the antibody binds NGF.
 9. The antibody of claim8, wherein the antibody further comprises a heavy chain variable region.10. An antibody comprising (a) a heavy chain variable region comprising:(i) a CDR1 region of SEQ ID NO:9, wherein I34 is substituted with S, L,V A, or I; and N35 is substituted with N, T or S; (ii) a CDR2 region ofSEQ ID NO:10, wherein M50 is I, G, Q, S, or L; A62 is A, or S; and L63is L or V; and (iii) a CDR3 region of SEQ ID NO:11, wherein Y100 is Y,L, or R; wherein Y101 is Y or W; wherein G103 is G, A, or S; whereinT104 is T or S; wherein S105 is S, A, or T; wherein Y106 is Y, R, T, orM; wherein Y107 is Y or F; wherein F108 is F or W; wherein D109 is D, N,or G; wherein Y110 is Y, K, S, R or T; and (b) a light chain variableregion comprising: (i) a CDR1 region of SEQ ID NO:12, wherein S26 is Sor F; D28 is D, S, A, or Y; and H32 is H, N, or Q; (ii) a CDR2 region ofSEQ ID NO:13, wherein 151 is I, T, V or A; and S56 is S or T; and (iii)a CDR3 region of SEQ ID NO:14, wherein S91 is S or E; K92 is K, H, R, orS; and wherein Y96 is Y or R; wherein the antibody binds NGF.
 11. Theantibody of any of claims 6-10, wherein the antibody binds human NGF.12. The antibody of claim 11, wherein the antibody further binds rodentNGF.
 13. The antibody of any of claims 6-10, wherein the antibody is amonoclonal antibody.
 14. The antibody of any of claims 6-10, wherein theantibody is a humanized antibody.
 15. The antibody of any of claims6-10, wherein the antibody binds NGF with a K_(D) of about 2 nM or less.16. The antibody of claim 15, wherein the K_(D) is about 100 pM or less.17. An antibody comprising a heavy chain variable region comprising: (a)a CDR1 region shown in SEQ ID NO: 3; (b) a CDR2 region shown in SEQ IDNO:4; and (c) a CDR3 region shown in SEQ ID NO:5; wherein the antibodybinds NGF.
 18. The antibody of claim 17, wherein the antibody furthercomprises a light chain variable region.
 19. An antibody comprising alight chain variable region comprising: (a) a CDR1 region shown in SEQID NO:6; (b) a CDR2 region shown in SEQ ID NO:7; and (c) a CDR3 regionshown in SEQ ID NO:8; wherein the antibody binds NGF.
 20. The antibodyof claim 19, wherein the antibody further comprises a heavy chainvariable region.
 21. The antibody of claim 19, wherein the antibodyfurther comprises a heavy chain variable region comprising: (a) a CDR1region shown in SEQ ID NO: 3; (b) a CDR2 region shown in SEQ ID NO:4;and (c) a CDR3 region shown in SEQ ID NO:5.
 22. The antibody of claim21, wherein the heavy chain variable region consists of the sequenceshown in SEQ ID NO:1.
 23. The antibody of claim 21, wherein the lightchain variable region consists of the sequence shown in SEQ ID NO:2. 24.The antibody of claim 22, wherein the heavy chain variable regionconsists of the sequence shown in SEQ ID NO:2.
 25. The antibody of claim21, wherein the heavy chain consists of the amino acid sequence shown inSEQ ID NO:16.
 26. The antibody of claim 21, wherein the light chainconsists of the amino acid sequence shown in SEQ ID NO:17.
 27. Theantibody of claim 25, wherein the light chain consists of the amino acidsequence shown in SEQ ID NO:17.
 28. The antibody of any of claims 17,19, or 21, wherein the antibody is humanized.
 29. The antibody of any ofclaims 17, 19, or 21, wherein the antibody is affinity matured.
 30. Theantibody of any of claims 17, 19, or 21, wherein the antibody is amonoclonal antibody.
 31. A pharmaceutical composition comprising (a) theantibody of any of claims 1, 6, 8, 10, 17, 19, 21, and 27, and (b) apharmaceutically acceptable excipient.
 32. A kit comprising the antibodyof any of claims 1, 6, 8, 10, 17, 19, 21, and
 27. 33. A method of makingthe antibody of claim 1, said method comprising expressing apolynucleotide encoding the antibody of claim 1 in a host cell.